Coxsackievirus B Exits the Host Cell in Shed Microvesicles Displaying Autophagosomal Markers

Coxsackievirus B3 (CVB3), a member of the picornavirus family and enterovirus genus, causes viral myocarditis, aseptic meningitis, and pancreatitis in humans. We genetically engineered a unique molecular marker, “fluorescent timer” protein, within our infectious CVB3 clone and isolated a high-titer recombinant viral stock (Timer-CVB3) following transfection in HeLa cells. “Fluorescent timer” protein undergoes slow conversion of fluorescence from green to red over time, and Timer-CVB3 can be utilized to track virus infection and dissemination in real time. Upon infection with Timer-CVB3, HeLa cells, neural progenitor and stem cells (NPSCs), and C2C12 myoblast cells slowly changed fluorescence from green to red over 72 hours as determined by fluorescence microscopy or flow cytometric analysis. The conversion of “fluorescent timer” protein in HeLa cells infected with Timer-CVB3 could be interrupted by fixation, suggesting that the fluorophore was stabilized by formaldehyde cross-linking reactions. Induction of a type I interferon response or ribavirin treatment reduced the progression of cell-to-cell virus spread in HeLa cells or NPSCs infected with Timer-CVB3. Time lapse photography of partially differentiated NPSCs infected with Timer-CVB3 revealed substantial intracellular membrane remodeling and the assembly of discrete virus replication organelles which changed fluorescence color in an asynchronous fashion within the cell. “Fluorescent timer” protein colocalized closely with viral 3A protein within virus replication organelles. Intriguingly, infection of partially differentiated NPSCs or C2C12 myoblast cells induced the release of abundant extracellular microvesicles (EMVs) containing matured “fluorescent timer” protein and infectious virus representing a novel route of virus dissemination. CVB3 virions were readily observed within purified EMVs by transmission electron microscopy, and infectious virus was identified within low-density isopycnic iodixanol gradient fractions consistent with membrane association. The preferential detection of the lipidated form of LC3 protein (LC3 II) in released EMVs harboring infectious virus suggests that the autophagy pathway plays a crucial role in microvesicle shedding and virus release, similar to a process previously described as autophagosome-mediated exit without lysis (AWOL) observed during poliovirus replication. Through the use of this novel recombinant virus which provides more dynamic information from static fluorescent images, we hope to gain a better understanding of CVB3 tropism, intracellular membrane reorganization, and virus-associated microvesicle dissemination within the host.     

Sequential Formation of Vaccinia Virus Proteins and Viral Deoxyribonucleic Acid Replication

In vaccinia virus-infected cell cultures, cellular protein synthesis was inhibited 50% at 2 hr postinfection (PI) and 80 to 90% by 4 hr PI. Input virus was responsible for this inhibition. Five early proteins, coded for by the viral genome, could be detected at 2 to 3 hr PI. Normally, their synthesis did not continue beyond 6 hr PI, at which time synthesis of a different set of proteins began. When DNA replication was blocked, synthesis of these early proteins continued until 9 to 12 hr PI. The bulk of the proteins which were incorporated into mature virus were synthesized at 8 hr PI and thereafter. The time of their formation was close to the time at which virus maturation occurred. However, 15% of the protein found in mature virus was synthesized early in the infectious cycle. The quantity of “early viral protein” which was not incorporated into mature virus was almost as large as the quantity of viral protein which did appear in mature virus. The “early” and “late” proteins could be shown to have separate and distinct immunological properties. The role of this large quantity of “early” protein is discussed.     

Analysis of Minimal Functions of Simian Virus 40 II. Enhancement of Oncogenic Transformation in Vitro by UV Irradiation

After light UV irradiation (5,000 to 10,000 ergs/mm²) “complete” and “defective” simian virus 40 (SV40) showed an enhancement of oncogenic transformation capacity in Syrian hamster kidney cells in vitro up to 180 and 270% of the controls, respectively. Simultaneously with the enhancement of transformation, an increase in T-antigen induction was observed in CV-1 cells infected with light UV-irradiated SV40; infectivity, however, was correspondingly reduced by 1 log₁₀. After strong UV irradiation (10,000 to 80,000 ergs/mm²) of “complete” and “defective” SV40, transformation capacity in vitro proved to be the most resistant viral function. It was only slightly reduced in comparison with a 4 to 5 log₁₀ reduction of infectivity. T-antigen induction of SV40 was also equally resistant to strong UV irradiation. We found no evidence of “multiplicity reactivation” involved in the high resistance of transformation capacity of SV40 after UV irradiation. Syrian hamster kidney cells transformed in vitro by UV-irradiated SV40 contained the SV40-specific T-antigen and showed the same morphology and growth characteristics as cells transformed by non-irradiated “complete” or “defective” SV40. They induced malignant tumors after subcutaneous inoculation into Syrian hamsters.     

Rescue of Simian Virus 40 from Cell Lines Transformed at High and at Low Input Multiplicities by Unirradiated or Ultraviolet-irradiated Virus

The relation between simian virus 40 (SV40) input multiplicity during transformation of primary mouse kidney cultures and the subsequent rescue of SV40 from clonal lines of transformed cells has been studied. Primary mouse kidney cultures were transformed with unirradiated SV40 at input multiplicities varying from 0.06 to 200 plaque-forming units (PFU) /cell or with SV40 irradiated with ultraviolet (UV) light to a survival of 0.04 to 0.01. All of the transformed lines contained the intranuclear SV40 T antigen, but cell-free extracts prepared from the transformed cell lines failed to yield infectious virus when assayed on monkey kidney cell (CV-1) monolayers. After fusion with susceptible CV-1 cells induced by UV-inactivated Sendai, all of the lines transformed by unirradiated virus yielded infectious SV40. The frequency of induction and the incidence of successful trials did not depend on the multiplicity of infection. “Good” yielders were obtained from mouse kidney cells transformed at the low input multiplicity of 0.06 PFU /cell. In contrast, only 4 of 12 clonal lines transformed at moderately low input multiplicity, and none of the lines transformed at very low input multiplicity with UV-irradiated virus yielded infectious SV40. The four positive lines have been classified as “poor” or “rare” yielders.     

Initiation of Vaccinia Virus Infection in Actinomycin D-pretreated Cells

The early steps in vaccinia virus infection were studied in HeLa cells which had been treated with actinomycin D (1 μg/ml) and then incubated for several hours in fresh medium prior to infection. Initiation of infection occurred in such cells even though the synthesis of cellular ribonucleic acid and deoxyribonucleic acid (DNA) was severely depressed. Thymidine kinase was synthesized in amounts that exceeded those found in untreated, infected cells. The breakdown of viral “cores” to liberate viral DNA and the synthesis of viral specific DNA-polymerase also occurred but were somewhat delayed. A deoxyribonuclease resembling an exonuclease was made by the infected, pretreated cells. The time course for these events suggested that the genetic code for synthesis of thymidine kinase can be expressed before “cores” are broken down, but the DNA-polymerase can be synthesized only after liberation of the viral DNA. The amount of viral specific DNA-polymerase which was made after infection was proportional to the total number of virus synthesizing sites even beyond the point where all the cells were infected with one infectious particle. A similar relationship was observed for the amount of thymidine kinase formed and for the rate of viral DNA synthesis from ³H-thymidine.     

Increased Early RNA Replication by Chimeric West Nile Virus W956IC Leads to IPS-1-Mediated Activation of NF-κB and Insufficient Virus-Mediated Counteraction of the Resulting Canonical Type I Interferon Signaling

Although infections with “natural” West Nile virus (WNV) and the chimeric W956IC WNV infectious clone virus produce comparable peak virus yields in type I interferon (IFN) response-deficient BHK cells, W956IC infection produces higher levels of “unprotected” viral RNA at early times after infection. Analysis of infections with these two viruses in IFN-competent cells showed that W956IC activated NF-κB, induced higher levels of IFN-β, and produced lower virus yields than WNV strain Eg101. IPS-1 was required for both increased induction of IFN-β and decreased yields of W956IC. In Eg101-infected cells, phospho-STAT1/STAT2 nuclear translocation was blocked at all times analyzed, while some phospho-STAT1/STAT2 nuclear translocation was still detected at 8 h after infection in W956IC-infected mouse embryonic fibroblasts (MEFs), and early viral protein levels were lower in these cells. A set of additional chimeras was made by replacing various W956IC gene regions with the Eg101 equivalents. As reported previously, for three of these chimeras, the low early RNA phenotype of Eg101 was restored in BHK cells. Analysis of infections with two of these chimeric viruses in MEFs detected lower early viral RNA levels, higher early viral protein levels, lower early IFN-β levels, and higher virus yields similar to those seen after Eg101 infection. The data suggest that replicase protein interactions directly or indirectly regulate genome switching between replication and translation at early times in favor of translation to minimize NF-κB activation and IFN induction by decreasing the amount of unprotected viral RNA, to produce sufficient viral protein to block canonical type I IFN signaling, and to efficiently remodel cell membranes for exponential genome amplification.     

Rare Branched Fatty Acids Characterize the Lipid Composition of the Intra-Aerobic Methane Oxidizer “Candidatus Methylomirabilis oxyfera”

The recently described bacterium “Candidatus Methylomirabilis oxyfera” couples the oxidation of the important greenhouse gas methane to the reduction of nitrite. The ecological significance of “Ca. Methylomirabilis oxyfera” is still underexplored, as our ability to identify the presence of this bacterium is thus far limited to DNA-based techniques. Here, we investigated the lipid composition of “Ca. Methylomirabilis oxyfera” to identify new, gene-independent biomarkers for the environmental detection of this bacterium. Multiple “Ca. Methylomirabilis oxyfera” enrichment cultures were investigated. In all cultures, the lipid profile was dominated up to 46% by the fatty acid (FA) 10-methylhexadecanoic acid (10MeC_16:0). Furthermore, a unique FA was identified that has not been reported elsewhere: the monounsaturated 10-methylhexadecenoic acid with a double bond at the Δ7 position (10MeC_16:1Δ7), which comprised up to 10% of the total FA profile. We propose that the typical branched fatty acids 10MeC_16:0 and 10MeC_16:1Δ7 are key and characteristic components of the lipid profile of “Ca. Methylomirabilis oxyfera.” The successful detection of these fatty acids in a peatland from which one of the enrichment cultures originated supports the potential of these unique lipids as biomarkers for the process of nitrite-dependent methane oxidation in the environment.     

Membrane Uncoating of Intact Enveloped Viruses

Experiments in the 1960s showed that Sendai virus, a paramyxovirus, fused its membrane with the host plasma membrane. After membrane fusion, the virus spontaneously “uncoated” with diffusion of the viral membrane proteins into the host plasma membrane and a merging of the host and viral membranes. This led to deposit of the viral ribonucleoprotein (RNP) and interior proteins in the cell cytoplasm. Later work showed that the common procedure then used to grow Sendai virus produced damaged, pleomorphic virions. Virions, which were grown under conditions that were not damaging, made a connecting structure between virus and cell at the region where the fusion occurred. The virus did not release its membrane proteins into the host membrane. The viral RNP was seen in the connecting structure in some cases. Uncoating of intact Sendai virus proceeds differently from uncoating described by the current standard model developed long ago with damaged virus. A model of intact paramyxovirus uncoating is presented and compared to what is known about the uncoating of other viruses.Enveloped virus entry at the plasma membrane includes binding of the virion to one or more receptors, changes in the virion components, membrane fusion, and membrane uncoating. The term “membrane uncoating” is being used to describe the separation of internal virion components from the viral membrane so the internal components can enter the cell. The term “uncoating” is sometimes used to mean the release of the viral genome from the capsid or other structures that have also entered the cell, but in this review, the term “membrane uncoating” will be used to represent only the separation of the virion internal contents and the viral envelope.Much of the original model of membrane fusion and uncoating was generally accepted as a result of a 1968 paper by Morgan and Howe (41). That paper provided strong evidence that Sendai virus (a paramyxovirus) entered a cell by fusion of the viral membrane with the cell plasma membrane. After membrane fusion, the virion rapidly lost its structure as the viral membrane merged with the host membrane and its components became part of the host membrane. The viral ribonucleoprotein (RNP) and internal proteins were released into the cytoplasm. This model of membrane uncoating is still generally accepted. For instance, in a 2007 virology text (24), this model was presented and illustrated with a figure from the Morgan and Howe paper. (The same figure is shown here as Fig. 2B.)Later, it was shown that Sendai viruses, which had been grown in fertilized chicken eggs, had different properties depending whether they had been harvested after growth for roughly 1 day (“early harvest”) or for several days (“late harvest”). The early-harvest viruses appear to be intact, but the late-harvest viruses have a different morphology and appear to be damaged (20, 26).This review summarizes data showing that intact early-harvest Sendai viruses uncoat quite differently from the way damaged late-harvest Sendai viruses uncoat. A model of intact paramyxovirus membrane uncoating is presented. The membrane uncoating of some other enveloped viruses that enter at the plasma membrane is compared to that described by this model.     

BeeDoctor,a Versatile MLPA-Based Diagnostic Tool for Screening Bee Viruses

The long-term decline of managed honeybee hives in the world has drawn significant attention to the scientific community and bee-keeping industry. A high pathogen load is believed to play a crucial role in this phenomenon, with the bee viruses being key players. Most of the currently characterized honeybee viruses (around twenty) are positive stranded RNA viruses. Techniques based on RNA signatures are widely used to determine the viral load in honeybee colonies. High throughput screening for viral loads necessitates the development of a multiplex polymerase chain reaction approach in which different viruses can be targeted simultaneously. A new multiparameter assay, called “BeeDoctor”, was developed based on multiplex-ligation probe dependent amplification (MLPA) technology. This assay detects 10 honeybee viruses in one reaction. “BeeDoctor” is also able to screen selectively for either the positive strand of the targeted RNA bee viruses or the negative strand, which is indicative for active viral replication. Due to its sensitivity and specificity, the MLPA assay is a useful tool for rapid diagnosis, pathogen characterization, and epidemiology of viruses in honeybee populations. “BeeDoctor” was used for screening 363 samples from apiaries located throughout Flanders; the northern half of Belgium. Using the “BeeDoctor”, virus infections were detected in almost eighty percent of the colonies, with deformed wing virus by far the most frequently detected virus and multiple virus infections were found in 26 percent of the colonies.     

Persistent Infections in L Cells with Temperature-Sensitive Mutants of Reovirus

Serial passage of reovirus temperature-sensitive (ts) mutant C(447) produced by passage 9 (P9) a heavily defective population of virus from which the double-stranded RNA genomic segments L₁, L₃, and M₁ were largely missing. Viral cores obtained from this P9 population were heterogeneous with respect to buoyant density in CsCl gradients, suggesting that particles were present with different combinations of deleted segments. Similar observations were made with the E(320) ts mutant of reovirus. By serial passage P15, 90% of the E(320) viral population was defective and the major missing genomic segments were L₁ and L₃. Persistent infections were readily established in monolayer cultures of L cells with P9 of C(447) virus and P15 of E(320) virus and in Vero cells with P9 of C(447) virus. Under similar conditions persistent infections could not be initiated with defective-free populations of C(447) or E(320) viruses. The greater the capacity of defective virus in the population to interfere with viral growth, the more readily persistent infection was initiated. During their maintenance persistently infected cells were subcultured approximately twice a week. More than 80% of the cells continuously produced virus. By subculture 6 the original ts infectious viral component had been replaced by a small-plaque mutant with a ts⁺ phenotype. Defective virus was always present in the carrier cells. In addition to the more commonly observed defectives whose cores banded at approximately ρ = 1.40 to 1.415 g/ml in CsCl gradients, a new class of defective core was seen banding in the region of 1.34 to 1.36 g/ml. This latter particle, which has not been thoroughly characterized as yet, is termed “light defective.” Persistently infected cells underwent periodic crises during their maintenance, during which the cultures partially lysed and then rapidly grew to confluence. Crises corresponded to a burst of infectious virus from the cells and a relatively low concentration of light defectives. During quiescent periods the concentration of light defectives amounted to as much as 98% of the total viral population. The function of light defectives is not yet clear, but it seems essential to assign major importance to defective virus in maintaining persistent infections in this system.     

Cells persistently infected with newcastle disease virus: I. Properties of mutants isolated from persistently infected L cells

The strain of Newcastle disease virus (NDV_pi) present in persistently infected L cells differed markedly from the Herts strain (NDV₀) used to initiate the infection. NDV_pi produced small plaques (less than 1 mm) in chick embryo cell cultures, whereas the wild type (NDV₀) produced large plaques (2 to 3 mm). The two viruses differed in a number of additional properties. Whereas 80% of adsorbed NDV₀ eluted from chicken red blood cells at 37 C, only about 20% of NDV_pi was recovered under similar conditions. There was no significant difference in the neuraminidase content of the two viruses. The infectivity of NDV₀ was stable for 1 hr at 48 C, whereas 99.9% of the infectivity of NDV_pi was destroyed. The two viruses also differed in lethality for chick embryos; NDV_pi had significantly reduced lethality for 9-day-old chick embryos when compared to NDV₀. In contrast to NDV₀, which produced an abortive infection in L cells, NDV_pi not only replicated effectively and destroyed these cells, but also induced significantly higher quantities of interferon than did NDV₀. These data furnished additional evidence for the lack of relationship of interferon production to abortive infection of L cells with NDV₀. In contrast, interferon was found to play a significant role in the maintenance of persistent infection.     

Destruction of L Cells by Mengo Virus: Use of Interferon to Study the Mechanism

Interferon, when added to L cells, inhibited the synthesis of infectious Mengo viral ribonucleic acid, hemagglutinins, and infectious virus by 85 to 95%. Serum-blocking antigens were also reduced by the action of interferon, but threefold excess amounts of these antigens accumulated in interferon-treated cultures above the amounts expected for the quantity of infectious virus that was produced in these cultures. Radioautographic analysis showed that 28 to 36% of the cells of an interferon-treated population synthesized viral ribonucleic acid and 36 to 47% produced viral antigens as determined by an immunofluorescence technique. Despite the reductions in synthesis of viral components, all cells in an interferon-treated culture underwent cytopathic effects at the same time as cells in infected cultures which had not been treated with interferon. The results are compatible with the hypothesis that the cell destruction which results from the infection of L cells with Mengo virus is due to a protein which is coded for by the virus but is not a component of the mature virion.     

Vaccines beyond antibodies: Spurred by pandemic research,are T‐cell vaccines moving closer to reality?

Lessons learned from the vaccines against SARS‐CoV‐2 has encouraged research and vaccine development aimed at mustering strong T cell responses against the pathogen. Subject Categories: Microbiology, Virology & Host Pathogen Interaction, Pharmacology & Drug Discovery

The new vaccines against SARS‐CoV‐2 elicited strong antibody responses in initial trials, which encouraged optimism amongst immunologists and public health experts who expected good efficacy. “With viral infections, it is almost unheard of to have a prophylactic vaccine that doesn’t work ultimately by generating neutralising antibody responses”, explained immunologist Kingston Mills at Trinity College Dublin in Ireland. However, the antibody response is not the whole story. “Efforts to explain how immunity is working against viruses to the general public has forced everyone to try to make things so simple that now what is left is a ridiculous oversimplified picture of the vertebrate immune system”, commented Antonio Bertoletti, infectious disease scientist at Duke‐National University of Singapore. In fact, there is increasing research focus on the role of T cells in mediating the cellular response to infections and how to stimulate these cells through vaccines.Antibodies work by recognising and attaching to surface structures of a virus or bacterium, which prevents the pathogen from infecting its target cells and mark it for destruction by other immune cells. However, pathogens can escape the antibody response via mutations that decrease the efficiency of antibodies from infection or vaccination. “You will still potentially get infected if you’re vaccinated, because the antibody response is not as strong as it was”, explained immunologist Luke O’Neill at Trinity College Dublin, Ireland. “But then the T cells will kick in and stop the virus when it is inside cells”. Simply put, antibodies tend to prevent infection, while T cells combat infection and illness. Specifically, CD4 helper T cells primarily encourage B cells to generate antibodies whereas CD8 “killer” T cells eliminate cancerous and virally infected cells.     

Gene-coding assignments of rotavirus double-stranded RNA segments 10 and 11

The gene-coding assignments for genome segments 10 and 11 of a simian virus and two human rotaviruses were determined. For those viruses having a “long” RNA gel pattern (electropherotype), segments 10 and 11 encoded proteins NS₃ and O₄, respectively. The human virus with a “short” electropherotype had the opposite assignments and also differed in (enzyme-linked immunosorbent assay) serotype from the human virus with a long electropherotype.     

Niche theory‐based modeling of assembly processes of viral communities in bats

Understanding the assembly processes of symbiont communities, including viromes and microbiomes, is important for improving predictions on symbionts’ biogeography and disease ecology. Here, we use phylogenetic, functional, and geographic filters to predict the similarity between symbiont communities, using as a test case the assembly process in viral communities of Mexican bats. We construct generalized linear models to predict viral community similarity, as measured by the Jaccard index, as a function of differences in host phylogeny, host functionality, and spatial co‐occurrence, evaluating the models using the Akaike information criterion. Two model classes are constructed: a “known” model, where virus–host relationships are based only on data reported in Mexico, and a “potential” model, where viral reports of all the Americas are used, but then applied only to bat species that are distributed in Mexico. Although the “known” model shows only weak dependence on any of the filters, the “potential” model highlights the importance of all three filter types—phylogeny, functional traits, and co‐occurrence—in the assemblage of viral communities. The differences between the “known” and “potential” models highlight the utility of modeling at different “scales” so as to compare and contrast known information at one scale to another one, where, for example, virus information associated with bats is much scarcer.     

Asymptomatic human CD4+ cytotoxic T-cell epitopes identified from herpes simplex virus glycoprotein B

The identification of “asymptomatic” (i.e., protective) epitopes recognized by T cells from herpes simplex virus (HSV)-seropositive healthy individuals is a prerequisite for an effective vaccine. Using the PepScan epitope mapping strategy, a library of 179 potential peptide epitopes (15-mers overlapping by 10 amino acids) was identified from HSV type 1 (HSV-1) glycoprotein B (gB), an antigen that induces protective immunity in both animal models and humans. Eighteen groups (G1 to G18) of 10 adjacent peptides each were first screened for T-cell antigenicity in 38 HSV-1-seropositive but HSV-2-seronegative individuals. Individual peptides within the two immunodominant groups (i.e., G4 and G14) were further screened with T cells from HLA-DR-genotyped and clinically defined symptomatic (n = 10) and asymptomatic (n = 10) HSV-1-seropositive healthy individuals. Peptides gB_161-175 and gB_166-180 within G4 and gB_661-675 within G14 recalled the strongest HLA-DR-dependent CD4⁺ T-cell proliferation and gamma interferon production. gB_166-180, gB_661-675, and gB_666-680 elicited ex vivo CD4⁺ cytotoxic T cells (CTLs) that lysed autologous HSV-1- and vaccinia virus (expressing gB)-infected lymphoblastoid cell lines. Interestingly, gB_166-180 and gB_666-680 peptide epitopes were strongly recognized by CD4⁺ T cells from 10 of 10 asymptomatic patients but not by CD4⁺ T cells from 10 of 10 symptomatic patients (P < 0.0001; analysis of variance posttest). Inversely, CD4⁺ T cells from symptomatic patients preferentially recognized gB_661-675 (P < 0.0001). Thus, we identified three previously unrecognized CD4⁺ CTL peptide epitopes in HSV-1 gB. Among these, gB_166-180 and gB_666-680 appear to be “asymptomatic” peptide epitopes and therefore should be considered in the design of future herpes vaccines.     

Transcription of Simian Virus 40 II. Hybridization of RNA Extracted from Different Lines of Transformed Cells to the Separated Strands of Simian Virus 40 DNA

The amount of simian virus 40 (SV40) DNA present in various SV40-transformed mouse cell lines and “revertants” isolated from them was determined. The number of viral DNA copies in the different cell lines ranged from 1.35 to 8.75 copies per diploid quantity of mouse cell DNA and from 2.2 to 14 copies per cell. The revertants had the same number of viral DNA copies per diploid quantity of mouse cell DNA as their parental cell lines. (However, they showed an increased number of viral DNA copies per cell due to their increased amount of DNA.) By using separated strands of SV40 DNA, the extent of each DNA strand transcribed into stable RNA species was determined for the transformed and “revertant” cell lines. From 30 to 80% of the “early” strand and from 0 to 20% of the “late” strand was present as stable RNA species in the cell lines tested. There was no alteration in the pattern of the stable viral RNA species present in three concanavalin A-selected revertants, whereas in a fluorodeoxyuridine-selected revertant there appeared to be less viral-specific RNA present in the cells.     

Anatomic Characteristics Associated with Head Splitting in Cabbage (Brassica oleracea var. capitata L.)

Cabbage belonging to Brassicaceae family is one of the most important vegetables cultivated worldwide. The economically important part of cabbage crop is head, formed by leaves which may be of splitting and non-splitting types. Cabbage varieties showing head splitting causes huge loss to the farmers and therefore finding the molecular and structural basis of splitting types would be helpful to breeders. To determine which anatomical characteristics were related to head-splitting in cabbage, we analyzed two contrasting cabbage lines and their offspring using a field emission scanning electron microscope. The inbred line “747” is an early head-splitting type, while the inbred line “748” is a head-splitting-resistant type. The petiole cells of “747” seems to be larger than those of “748” at maturity; however, there was no significant difference in petiole cell size at both pre-heading and maturity stages. The lower epidermis cells of “747” were larger than those of “748” at the pre-heading and maturity stages. “747” had thinner epidermis cell wall than “748” at maturity stage, however, there was no difference of the epidermis cell wall thickness in the two lines at the pre-heading stage. The head-splitting plants in the F₁ and F₂ population inherited the larger cell size and thinner cell walls of epidermis cells in the petiole. In the petiole cell walls of “747” and the F₁ and F₂ plants that formed splitting heads, the cellulose microfibrils were loose and had separated from each other. These findings verified that anomalous cellulose microfibrils, larger cell size and thinner-walled epidermis cells are important genetic factors that make cabbage heads prone to splitting.     

Paleo-Immunology: Evidence Consistent with Insertion of a Primordial Herpes Virus-Like Element in the Origins of Acquired Immunity

Background

The RAG encoded proteins, RAG-1 and RAG-2 regulate site-specific recombination events in somatic immune B- and T-lymphocytes to generate the acquired immune repertoire. Catalytic activities of the RAG proteins are related to the recombinase functions of a pre-existing mobile DNA element in the DDE recombinase/RNAse H family, sometimes termed the “RAG transposon”.

Methodology/Principal Findings

Novel to this work is the suggestion that the DDE recombinase responsible for the origins of acquired immunity was encoded by a primordial herpes virus, rather than a “RAG transposon.” A subsequent “arms race” between immunity to herpes infection and the immune system obscured primary amino acid similarities between herpes and immune system proteins but preserved regulatory, structural and functional similarities between the respective recombinase proteins. In support of this hypothesis, evidence is reviewed from previous published data that a modern herpes virus protein family with properties of a viral recombinase is co-regulated with both RAG-1 and RAG-2 by closely linked cis-acting co-regulatory sequences. Structural and functional similarity is also reviewed between the putative herpes recombinase and both DDE site of the RAG-1 protein and another DDE/RNAse H family nuclease, the Argonaute protein component of RISC (RNA induced silencing complex).

Conclusions/Significance

A “co-regulatory” model of the origins of V(D)J recombination and the acquired immune system can account for the observed linked genomic structure of RAG-1 and RAG-2 in non-vertebrate organisms such as the sea urchin that lack an acquired immune system and V(D)J recombination. Initially the regulated expression of a viral recombinase in immune cells may have been positively selected by its ability to stimulate innate immunity to herpes virus infection rather than V(D)J recombination Unlike the “RAG-transposon” hypothesis, the proposed model can be readily tested by comparative functional analysis of herpes virus replication and V(D)J recombination.