Loss of cytoskeletal transport during egress critically attenuates ectromelia virus infection in vivo

Egress of wrapped virus (WV) to the cell periphery following vaccinia virus (VACV) replication is dependent on interactions with the microtubule motor complex kinesin-1 and is mediated by the viral envelope protein A36. Here we report that ectromelia virus (ECTV), a related orthopoxvirus and the causative agent of mousepox, encodes an A36 homologue (ECTV-Mos-142) that is highly conserved despite a large truncation at the C terminus. Deleting the ECTV A36R gene leads to a reduction in the number of extracellular viruses formed and to a reduced plaque size, consistent with a role in microtubule transport. We also observed a complete loss of virus-associated actin comets, another phenotype dependent on A36 expression during VACV infection. ECTV ΔA36R was severely attenuated when used to infect the normally susceptible BALB/c mouse strain. ECTV ΔA36R replication and spread from the draining lymph nodes to the liver and spleen were significantly reduced in BALB/c mice and in Rag-1-deficient mice, which lack T and B lymphocytes. The dramatic reduction in ECTV ΔA36R titers early during the course of infection was not associated with an augmented immune response. Taken together, these findings demonstrate the critical role that subcellular transport pathways play not only in orthopoxvirus infection in an in vitro context but also during orthopoxvirus pathogenesis in a natural host. Furthermore, despite the attenuation of the mutant virus, we found that infection nonetheless induced protective immunity in mice, suggesting that orthopoxvirus vectors with A36 deletions may be considered another safe vaccine alternative.     

Western Palaearctic phylogeography of an inquiline oak gall wasp,Synergus umbraculus

Insect‐induced galls on plants comprise species‐rich but self‐contained communities of herbivores and natural enemies. In the present study, we focus on galls induced by cynipid gall wasps on oaks, and on the least‐known trophic level that these galls contain: inquilines. These insects, also cynipids, feed on gall tissue and are an abundant but taxonomically poorly understood part of an otherwise well‐studied system. We used DNA sequence data to examine spatial patterns in the genetic diversity of Synergus umbraculus Olivier 1791 (Hymenoptera: Cynipidae: Synergini), a widespread species attacking many host galls across the Western Palaearctic. Analysis of 239 cytochrome b sequences revealed eight haplogroups showing significant phylogeographic pattern across the Western Palaearctic, corresponding to putative glacial refugia in Iberia, Central Europe, Turkey, and Iran. There were significant genetic discontinuities across the Pyrenees and the Anatolian diagonal but no impact of the Alps, suggesting that significant discontinuities have biotic rather than physical causes. Detailed analysis of sites in the Carpathian Basin reveal a high diversity and low spatial structure, and identify Central Europe as the source of colonists for Quaternary colonization of Germany, France, and Britain. We found no evidence for host‐associated differentiation of S. umbraculus lineages associated with the most common cynipid host galls, suggesting frequent shifts within the host gall assemblage by inquiline lineages. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102 , 750–764.     

Are we ready for therapeutic drug monitoring of biologic therapeutics?

In the previous issue of Arthritis Research & Therapy, Ducourau and colleagues report that they retrospectively detected anti-infliximab antibodies in 21% of patients with rheumatic diseases. Patients with anti-infliximab antibodies had lower serum drug concentrations. These findings contribute to the existing evidence of immunogenicity of biologicals and its clinical relevance. We argue for therapeutic drug monitoring to optimize treatment response.     

A36-dependent Actin Filament Nucleation Promotes Release of Vaccinia Virus

Cell-to-cell transmission of vaccinia virus can be mediated by enveloped virions that remain attached to the outer surface of the cell or those released into the medium. During egress, the outer membrane of the double-enveloped virus fuses with the plasma membrane leaving extracellular virus attached to the cell surface via viral envelope proteins. Here we report that F-actin nucleation by the viral protein A36 promotes the disengagement of virus attachment and release of enveloped virus. Cells infected with the A36^YdF virus, which has mutations at two critical tyrosine residues abrogating localised actin nucleation, displayed a 10-fold reduction in virus release. We examined A36^YdF infected cells by transmission electron microscopy and observed that during release, virus appeared trapped in small invaginations at the plasma membrane. To further characterise the mechanism by which actin nucleation drives the dissociation of enveloped virus from the cell surface, we examined recombinant viruses by super-resolution microscopy. Fluorescently-tagged A36 was visualised at sub-viral resolution to image cell-virus attachment in mutant and parental backgrounds. We confirmed that A36^YdF extracellular virus remained closely associated to the plasma membrane in small membrane pits. Virus-induced actin nucleation reduced the extent of association, thereby promoting the untethering of virus from the cell surface. Virus release can be enhanced via a point mutation in the luminal region of B5 (P189S), another virus envelope protein. We found that the B5^P189S mutation led to reduced contact between extracellular virus and the host membrane during release, even in the absence of virus-induced actin nucleation. Our results posit that during release virus is tightly tethered to the host cell through interactions mediated by viral envelope proteins. Untethering of virus into the surrounding extracellular space requires these interactions be relieved, either through the force of actin nucleation or by mutations in luminal proteins that weaken these interactions.     

Population and genetic outcomes 20 years after reintroducing bobcats (Lynx rufus) to Cumberland Island,Georgia USA

In 1988–1989, 32 bobcats Lynx rufus were reintroduced to Cumberland Island (CUIS), Georgia, USA, from which they had previously been extirpated. They were monitored intensively for 3 years immediately post‐reintroduction, but no estimation of the size or genetic diversity of the population had been conducted in over 20 years since reintroduction. We returned to CUIS in 2012 to estimate abundance and effective population size of the present‐day population, as well as to quantify genetic diversity and inbreeding. We amplified 12 nuclear microsatellite loci from DNA isolated from scats to establish genetic profiles to identify individuals. We used spatially explicit capture–recapture population estimation to estimate abundance. From nine unique genetic profiles, we estimate a population size of 14.4 (SE = 3.052) bobcats, with an effective population size (N _e) of 5–8 breeding individuals. This is consistent with predictions of a population viability analysis conducted at the time of reintroduction, which estimated the population would average 12–13 bobcats after 10 years. We identified several pairs of related bobcats (parent‐offspring and full siblings), but ~75% of the pairwise comparisons were typical of unrelated individuals, and only one individual appeared inbred. Despite the small population size and other indications that it has likely experienced a genetic bottleneck, levels of genetic diversity in the CUIS bobcat population remain high compared to other mammalian carnivores. The reintroduction of bobcats to CUIS provides an opportunity to study changes in genetic diversity in an insular population without risk to this common species. Opportunities for natural immigration to the island are limited; therefore, continued monitoring and supplemental bobcat reintroductions could be used to evaluate the effect of different management strategies to maintain genetic diversity and population viability. The successful reintroduction and maintenance of a bobcat population on CUIS illustrates the suitability of translocation as a management tool for re‐establishing felid populations.     

Systematic re‐appraisal of the gall‐usurping wasp genus Synophrus Hartig, 1843 (Hymenoptera: Cynipidae: Synergini)

Several unanswered questions remain regarding the taxonomy and phylogeny of inquiline gallwasps (Cynipidae: Synergini), obligate inhabitants of plant galls induced primarily by other gallwasps (Cynipidae: Cynipini and Diplolepidini). Here we use morphological and molecular data to revise the inquiline genus Synophrus, members of which are notable for extensively modifying the structure of galls induced by oak gallwasp hosts on oaks in the section Cerris of Quercus subgenus Quercus in the Western Palaearctic. Previous taxonomic treatments have recognized three Western Palaearctic species of Synophrus: S. pilulae, S. politus and S. olivieri. Our results support the establishment of four additional Western Palaearctic species: Synophrus hungaricus sp.n. , S. libani sp.n. , S. syriacus sp.n. and S. hispanicus sp.n. We describe and diagnose these new taxa, analyse their phylogenetic relationships, and show that Synophrus inquilines are able to impose their own gall phenotypes on those of their hosts. We provide an updated key to Synophrus.     

Mimicry in recognition of cardiac myosin peptides by heart-intralesional T cell clones from rheumatic heart disease

Molecular mimicry between Streptococcus pyogenes Ags and human proteins has been considered as a mechanism leading to autoimmune reactions in rheumatic fever and rheumatic heart disease (RHD). Cardiac myosin has been shown as a putative autoantigen recognized by autoantibodies of rheumatic fever patients. We assessed the human heart-intralesional T cell response against human light meromyosin (LMM) and streptococcal M5 peptides and mitral-valve-derived proteins by proliferation assay. Cytokines induced by LMM peptides were also evaluated. The frequency of intralesional T cell clones that recognized LMM peptides was 63.2%. Thirty-four percent of T cell clones presented cross-reactivity with different patterns: 1) myosin and valve-derived proteins; 2) myosin and streptococcal M5 peptides; and 3) myosin, valve-derived proteins and M5 peptides. In addition, several LMM peptides were recognized simultaneously showing a multiple reactivity pattern of heart-infiltrating T cells. Inflammatory cytokines (IFN-gamma and TNF-alpha) were predominantly produced by heart-infiltrating T cells upon stimulation with LMM peptides. The alignment of LMM and streptococcal M5 peptides showed frequent homology among conserved amino acid substitutions. This is the first study showing the cellular response by human heart-infiltrating T cells against cardiac myosin epitopes in RHD patients. The high percentage of reactivity against cardiac myosin strengthens its role as one of the major autoantigens involved in rheumatic heart lesions. T cell reactivity toward myosin epitopes in RHD patients may also trigger the broad recognition of valvular proteins with structural or functional similarities.     

Identification of host cell proteins which interact with herpes simplex virus type 1 tegument protein pUL37

The herpes simplex virus type 1 (HSV-1) structural tegument protein pUL37, which is conserved across the Herpesviridae family, is known to be essential for secondary envelopment during the egress of viral particles. To shed light on additional roles of pUL37 during viral replication a yeast two-hybrid screen of a human brain cDNA library was undertaken. This screen identified ten host cell proteins as potential pUL37 interactors. One of the interactors, serine threonine kinase TAOK3, was subsequently confirmed to interact with pUL37 using an in vitro pulldown assay. Such host cell/pUL37 interactions provide further insights into the multifunctional role of this herpesviral tegument protein.