A beta-herpesvirus with fluorescent capsids to study transport in living cells.

Fluorescent tagging of viral particles by genetic means enables the study of virus dynamics in living cells. However, the study of beta-herpesvirus entry and morphogenesis by this method is currently limited. This is due to the lack of replication competent, capsid-tagged fluorescent viruses. Here, we report on viable recombinant MCMVs carrying ectopic insertions of the small capsid protein (SCP) fused to fluorescent proteins (FPs). The FPs were inserted into an internal position which allowed the production of viable, fluorescently labeled cytomegaloviruses, which replicated with wild type kinetics in cell culture. Fluorescent particles were readily detectable by several methods. Moreover, in a spread assay, labeled capsids accumulated around the nucleus of the newly infected cells without any detectable viral gene expression suggesting normal entry and particle trafficking. These recombinants were used to record particle dynamics by live-cell microscopy during MCMV egress with high spatial as well as temporal resolution. From the resulting tracks we obtained not only mean track velocities but also their mean square displacements and diffusion coefficients. With this key information, we were able to describe particle behavior at high detail and discriminate between particle tracks exhibiting directed movement and tracks in which particles exhibited free or anomalous diffusion

Randomized clinical trial to evaluate the effects of a prepartum cholecalciferol injection on postpartum serum calcium dynamics and health and performance in early-lactation multiparous dairy cows

The objectives of the present study were (1) to evaluate the effect of prepartum cholecalciferol treatment on serum Ca concentration during the first 10 d after calving and (2) to evaluate the effect of treatment on subsequent health and performance. Multiparous Holstein cows (n = 377) from one dairy farm were fed a negative dietary cation-anion difference diet (−31 mEq/kg of DM) for the last 21 d of gestation. On d 275, the animals were randomly assigned to a control or a treatment group. Cows in the control group were left untreated, and cows in the treatment group received an injection of 12 × 106 IU of cholecalciferol intramuscularly on the day of enrollment. If treated cows did not deliver the calf within 6 d, they were reinjected with 10 × 106 IU of cholecalciferol. Blood samples were drawn on 1, 2, 3, 5, 7, and 10 days in milk (DIM) and analyzed for serum Ca, P, and Mg concentrations. In a subsample of cows (50 control cows, 35 cows treated once with cholecalciferol, and 15 cows treated twice) serum haptoglobin, nonesterified fatty acids, β-hydroxybutyrate, and 25-hydroxycholecalciferol concentrations were analyzed on 1, 5, and 10 DIM. Binary data [retained placenta (RP), metritis] were analyzed using logistic regression models. Repeated measures ANOVA with first-order autoregressive covariance was performed to evaluate the treatment effect on milk yield over the first 10 test days after parturition, 25-hydroxycholecalciferol, serum Ca, P, Mg, β-hydroxybutyrate, nonesterified fatty acids, and haptoglobin concentrations. Cox proportional hazards were used to model the time to event outcomes (time to pregnancy within 200 d, culling until 300 DIM). After enrollment of 31.4% of cows and a preliminary analysis, adverse reactions became apparent, and the study was stopped. Cows treated with cholecalciferol had a greater risk of incurring RP and metritis. The adjusted mean incidences were 2.0%, 7.7%, and 4.0% for RP, and 21.6%, 39.3%, and 33.3% for metritis for control cows, cows treated once, and cows treated twice with cholecalciferol, respectively. Compared with control cows, cows injected once with 12 × 106 IU of cholecalciferol produced less energy-corrected milk on the first (−3.76 kg) and second (−2.75 kg) test days, respectively. Cows injected twice with cholecalciferol (12 × 106 IU of cholecalciferol and 10 × 106 IU 1 wk later) had a reduced milk yield only at first test day (−3.80 kg). Treatment with cholecalciferol led to a significant increase in 25-hydroxycholecalciferol on d 1, 5, and 10 after calving. Serum Ca and P concentrations were significantly increased in cows treated with cholecalciferol, but serum Mg concentrations were significantly reduced. Haptoglobin concentrations were significantly increased on 5 DIM in cows injected once with 12 × 106 IU of cholecalciferol. Although we observed no effect of treatment on culling until 300 DIM, time to pregnancy was delayed by 34 d in cows injected once with 12 × 106 IU of cholecalciferol. In the present study, injection with 12 × 106 IU of cholecalciferol had detrimental effects on health and milk production despite the beneficial effects on Ca homeostasis

The Herpes Simplex Virus Protein pUL31 Escorts Nucleocapsids to Sites of Nuclear Egress, a Process Coordinated by Its N-Terminal Domain

Progeny capsids of herpesviruses leave the nucleus by budding through the nuclear envelope. Two viral proteins, the membrane protein pUL34 and the nucleo-phosphoprotein pUL31 form the nuclear egress complex that is required for capsid egress out of the nucleus. All pUL31 orthologs are composed of a diverse N-terminal domain with 1 to 3 basic patches and a conserved C-terminal domain. To decipher the functions of the N-terminal domain, we have generated several Herpes simplex virus mutants and show here that the N-terminal domain of pUL31 is essential with basic patches being critical for viral propagation. pUL31 and pUL34 entered the nucleus independently of each other via separate routes and the N-terminal domain of pUL31 was required to prevent their premature interaction in the cytoplasm. Unexpectedly, a classical bipartite nuclear localization signal embedded in this domain was not required for nuclear import of pUL31. In the nucleus, pUL31 associated with the nuclear envelope and newly formed capsids. Viral herpesviralmutants lacking the N-terminal domain or with its basic patches neutralized still associated with nucleocapsids but were unable to translocate them to the nuclear envelope. Replacing the authentic basic patches with a novel artificial one resulted in HSV1(17(+)) Lox-UL31-hbpmp1mp2, that was viable but delayed in nuclear egress and compromised in viral production. Thus, while the C-terminal domain of pUL31 is sufficient for the interaction with nucleocapsids, the N-terminal domain was essential for capsid translocation to sites of nuclear egress and a coordinated interaction with pUL34. Our data indicate an orchestrated sequence of events with pUL31 binding to nucleocapsids and escorting them to the inner nuclear envelope. We propose a common mechanism for herpesviral nuclear egress: pUL31 is required for intranuclear translocation of nucleocapsids and subsequent interaction with pUL34 thereby coupling capsid maturation with primary envelopment

Varicella zoster virus glycoprotein C increases chemokine-mediated leukocyte migration

Varicella zoster virus (VZV) is a highly prevalent human pathogen that establishes latency in neurons of the peripheral nervous system. Primary infection causes varicella whereas reactivation results in zoster, which is often followed by chronic pain in adults. Following infection of epithelial cells in the respiratory tract, VZV spreads within the host by hijacking leukocytes, including T cells, in the tonsils and other regional lymph nodes, and modifying their activity. In spite of its importance in pathogenesis, the mechanism of dissemination remains poorly understood. Here we addressed the influence of VZV on leukocyte migration and found that the purified recombinant soluble ectodomain of VZV glycoprotein C (rSgC) binds chemokines with high affinity. Functional experiments show that VZV rSgC potentiates chemokine activity, enhancing the migration of monocyte and T cell lines and, most importantly, human tonsillar leukocytes at low chemokine concentrations. Binding and potentiation of chemokine activity occurs through the C-terminal part of gC ectodomain, containing predicted immunoglobulin-like domains. The mechanism of action of VZV rSgC requires interaction with the chemokine and signalling through the chemokine receptor. Finally, we show that VZV viral particles enhance chemokine-dependent T cell migration and that gC is partially required for this activity. We propose that VZV gC activity facilitates the recruitment and subsequent infection of leukocytes and thereby enhances VZ

Varicella zoster virus glycoprotein C increases chemokine-mediated leukocyte migration.

Varicella zoster virus (VZV) is a highly prevalent human pathogen that establishes latency in neurons of the peripheral nervous system. Primary infection causes varicella whereas reactivation results in zoster, which is often followed by chronic pain in adults. Following infection of epithelial cells in the respiratory tract, VZV spreads within the host by hijacking leukocytes, including T cells, in the tonsils and other regional lymph nodes, and modifying their activity. In spite of its importance in pathogenesis, the mechanism of dissemination remains poorly understood. Here we addressed the influence of VZV on leukocyte migration and found that the purified recombinant soluble ectodomain of VZV glycoprotein C (rSgC) binds chemokines with high affinity. Functional experiments show that VZV rSgC potentiates chemokine activity, enhancing the migration of monocyte and T cell lines and, most importantly, human tonsillar leukocytes at low chemokine concentrations. Binding and potentiation of chemokine activity occurs through the C-terminal part of gC ectodomain, containing predicted immunoglobulin-like domains. The mechanism of action of VZV rSgC requires interaction with the chemokine and signalling through the chemokine receptor. Finally, we show that VZV viral particles enhance chemokine-dependent T cell migration and that gC is partially required for this activity. We propose that VZV gC activity facilitates the recruitment and subsequent infection of leukocytes and thereby enhances VZV systemic dissemination in humans

Varicella zoster virus glycoprotein C increases chemokine-mediated leukocyte migration

Varicella zoster virus (VZV) is a highly prevalent human pathogen that establishes latency in neurons of the peripheral nervous system. Primary infection causes varicella whereas reactivation results in zoster, which is often followed by chronic pain in adults. Following infection of epithelial cells in the respiratory tract, VZV spreads within the host by hijacking leukocytes, including T cells, in the tonsils and other regional lymph nodes, and modifying their activity. In spite of its importance in pathogenesis, the mechanism of dissemination remains poorly understood. Here we addressed the influence of VZV on leukocyte migration and found that the purified recombinant soluble ectodomain of VZV glycoprotein C (rSgC) binds chemokines with high affinity. Functional experiments show that VZV rSgC potentiates chemokine activity, enhancing the migration of monocyte and T cell lines and, most importantly, human tonsillar leukocytes at low chemokine concentrations. Binding and potentiation of chemokine activity occurs through the C-terminal part of gC ectodomain, containing predicted immunoglobulin-like domains. The mechanism of action of VZV rSgC requires interaction with the chemokine and signalling through the chemokine receptor. Finally, we show that VZV viral particles enhance chemokine-dependent T cell migration and that gC is partially required for this activity. We propose that VZV gC activity facilitates the recruitment and subsequent infection of leukocytes and thereby enhances VZV systemic dissemination in humans

Quantification of nuclear aggregate formation.

<p>Vero cells were infected with 10 PFU/cell of HSV1(17⁺)blueLox (B) or HSV1(17⁺)blueLox-GFPVP26_{Δaa<b>1</b>–7}-gDmRFP (C), and the cells were fixed at 4, 6, 8, 10, or 12 h. After permeabilization, HSV1(17⁺)blueLox infected cells were labeled with an antibody directed against VP26. According to their intranuclear VP26 phenotype, the cells were classified into “none”, “single”, “grainy” and “aggregate” (A). The numbers above the columns describe the number of nuclei analyzed for each time point.</p

HSV1-VP26 constructs.

<p>(A) 1^st column: HSV1 constructs in which the SCP VP26 has been tagged with different fluorescent protein domains. 2^nd column: Genomic organization of the UL35 region approximately drawn to scale. The gene UL35 coding for VP26 has been disrupted by replacing it with lacZ or an rpsLneo cassette out of frame. Some constructs lack a 65 bp region upstream of UL35 (D65 bp) including the first seven N-terminal codons of VP26 (Daa1–7), while others lack only four (Δaa1–4) or just one (Daa1) codon. For the present study, the fluorescent protein tag was inserted between VP26 residues 4 and 8 (Daa5–7). Due to the mutagenesis, some strains contain additional linkers (*, AW; **, NSS; ***, HST). 3^rd column: Propensity of the fluorescent protein (FP) to dimerize <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044177#pone.0044177-Espagne1" target="_blank">[76]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044177#pone.0044177-Campbell1" target="_blank">[80]</a>. 4^th column: Ability of the construct to replicate and to form plaques (+++, similar to wild type; ++ attenuated, but robust growth; − strongly attenuated, tiny plaques; −−, single fluorescent cells, no plaques). 5^th column: Propensity of the construct to induce nuclear aggregates (+++, large irregular shaped aggregates; ++, large aggregates early after infection or transfection; +, aggregates late in infection; − aggregates in less than 2% of cells even late in infection). 6^th column: References. (B) Nucleotide (upper lines) and amino acid (lower lines) sequences of the UL34/UL35 (pUL34/VP26) intergenic region. The 3¢ end of the UL34 ORF until the 5¢ start of the UL35 ORF are shown for wild type HSV-1, the GFPVP26_{Äaa<b>5</b>–7} (Äaa5–7) and GFPVP26_{Äaa<b>1</b>–7} (Äaa1–7) mutants. Additional nucleotides inserted during mutagenesis are shown in bold capitals, and the GFP amino acids are shown in italics. Putative Inr late promoter elements are underlined with the element perfectly matching the consensus sequence being underlined and in italics. The original amino acids encoded by UL35 are shown in bold capitals, the inserted GFP residues in italic capitals and the additional linker residues in normal script capitals.</p