Quiescent Infection Research Articles

Potential of fumigation of orange fruits with volatile organic compounds produced by Saccharomyces cerevisiae to control citrus black spot disease at postharvest

Volatile organic compounds (VOCs) from microbial origin are relevant in biological interactions and are considered promising environmentally safer fumigant agents to control postharvest diseases of fruits. The antagonist yeast Saccharomyces cerevisiae produces VOCs able to inhibit the development of plant pathogens, including the filamentous fungus Phyllosticta citricarpa, causal agent of citrus black spot. Thus, it was evaluated the effectiveness of VOCs produced by S. cerevisiae to control P. citricarpa in orange fruits stored in sealed glass containers. The exposure of P. citricarpa growing on potato-dextrose-agar to the synthetic mixture of VOCs, originally identified from S. cerevisiae, affected negatively the phytopathogen. Individually, 3-methyl-1-butanol and 2-methyl-1-butanol were the most effective VOCs inhibiting completely the mycelial growth and the germination and appressorium formation by conidia. Seven-day fumigation of orange fruits carrying quiescent infections of P. citricarpa, employing the VOC-producing S. cerevisiae or 3-methyl-1-butanol at 0.33μlml−1 of air space, controlled the development of new lesions close to 90%, even after removing the fruits from the VOC influence. Therefore, the biological fumigation of citrus fruits with S. cerevisiae or the use of formulations based on their VOCs are promising ecofriendly approaches to control citrus black spot during storage and shipment.

Transformation of Cells by Photoinactivated Murine Gamma-Herpesvirus 68 during Nonproductive and Quiescent Infection

Infection of human MRC-5 cells and mouse NIH-3T3 cells with a murine gamma-herpesvirus (MuHV-4 strain 68; MHV-68) photoinactivated by visible light in the presence of methylene blue (MB) resulted in nonproductive infection and the appearance of morphologically transformed cells. Two stably transformed cell lines were derived from both of these cell types and were confirmed to contain both viral DNA and antigen. Next, a quiescent MHV-68 infection in MRC-5 and NIH-3T3 cells was established after cultivation at 41°C in the presence of phosphonoacetic acid. Following the exposure of quiescently infected cells to visible light for 120 s (5 times daily for 6 days) in the presence of MB, both MRC-5 and NIH-3T3 cells were observed to acquire transformed phenotypes. The cytopathic effect was observed in cells after 4-5 passages, after which the cells degenerated. However, when human interferon (IFN)-α and mouse IFN-β were added to the media of quiescently infected MRC-5 and NIH-3T3 cells during the photoinactivating procedure, 2 stable transformed cell lines containing both viral DNA and the antigen were obtained and resembled those attained following nonproductive infection with photoinactivated virus.

Hepatitis C in Pregnancy: Screening and Current Treatments

Hepatitis C virus (HCV) infection affects around 170 million men and women all over the world. Many patients with chronic HCV infection do not present with active symptoms until years after contracting the disease. In particular, women with quiescent HCV infection may become pregnant and deliver their child without knowing they were even infected. Thus, vertical transmission of HCV from mother to infant remains the single largest contributor to childhood HCV infection. Specific factors present before, during, or after pregnancy can increase the risk of HCV transmission from mother to infant. Some of these factors include a high viral load at the time of delivery, HIV co-infection, vaginal or perineal lacerations during delivery, and/or having a prolonged rupture of membranes. Despite these specific instances, the transmission rate from mother to infant still remains fairly low at around 1–8 %. New medications in the category of direct-acting antivirals may show promise in treating HCV in pregnant women. Many of these oral drug combinations such as sofosbuvir/ledipasvir, elbasvir/grazoprevir, and ombitasvir/paritaprevir/ritonavir/dasabuvir have been studied in pregnant animal populations but have limited to no results in human populations. The door remains open for future studies of these drugs to treat pregnant women during pregnancy that may help reduce viral loads and prevent vertical transmission of HCV infection to the infant.

Dual-Color Herpesvirus Capsids Discriminate Inoculum from Progeny and Reveal Axonal Transport Dynamics.

Alphaherpesviruses such as herpes simplex virus and pseudorabies virus (PRV) are neuroinvasive double-stranded DNA (dsDNA) viruses that establish lifelong latency in peripheral nervous system (PNS) neurons of their native hosts. Following reactivation, infection can spread back to the initial mucosal site of infection or, in rare cases, to the central nervous system, with usually serious outcomes. During entry and egress, viral capsids depend on microtubule-based molecular motors for efficient and fast transport. In axons of PNS neurons, cytoplasmic dynein provides force for retrograde movements toward the soma, and kinesins move cargo in the opposite, anterograde direction. The dynamic properties of virus particles in cells can be imaged by fluorescent protein fusions to the small capsid protein VP26, which are incorporated into capsids. However, single-color fluorescent protein tags fail to distinguish the virus inoculum from progeny. Therefore, we established a dual-color system by growing a recombinant PRV expressing a red fluorescent VP26 fusion (PRV180) on a stable cell line expressing a green VP26 fusion (PK15-mNG-VP26). The resulting dual-color virus preparation (PRV180G) contains capsids tagged with both red and green fluorescent proteins, and 97% of particles contain detectable levels of mNeonGreen (mNG)-tagged VP26. After replication in neuronal cells, all PRV180G progeny exclusively contain monomeric red fluorescent protein (mRFP)-VP26-tagged capsids. We used PRV180G for an analysis of axonal capsid transport dynamics in PNS neurons. Fast dual-color total internal reflection fluorescence (TIRF) microscopy, single-particle tracking, and motility analyses reveal robust, bidirectional capsid motility mediated by cytoplasmic dynein and kinesin during entry, whereas egressing progeny particles are transported exclusively by kinesins. IMPORTANCE Alphaherpesviruses are neuroinvasive viruses that infect the peripheral nervous system (PNS) of infected hosts as an integral part of their life cycle. Establishment of a quiescent or latent infection in PNS neurons is a hallmark of most alphaherpesviruses. Spread of infection to the central nervous system is surprisingly rare in natural hosts but can be fatal. Pseudorabies virus (PRV) is a broad-host-range swine alphaherpesvirus that enters neuronal cells and utilizes intracellular transport processes to establish infection and to spread between cells. By using a virus preparation with fluorescent viral capsids that change color depending on the stage of the infectious cycle, we find that during entry, axons of PNS neurons support robust, bidirectional capsid motility, similar to cellular cargo, toward the cell body. In contrast, progeny particles appear to be transported unidirectionally by kinesin motors toward distal egress sites.

Friend or foe? Biological and ecological traits of the European ash dieback pathogen Hymenoscyphus fraxineus in its native environment.

Hymenoscyphus fraxineus, an introduced ascomycete fungus and primary causal agent of European ash dieback, was investigated on Fraxinus mandshurica trees in its native range in Primorye region of Far East Russia. This evidence is the first report of H. fraxineus on healthy, asymptomatic F. mandshurica trees. High-throughput sequencing revealed 49 distinct fungal taxa associated with leaves of F. mandshurica, 12 of which were identified to species level. Phyllosphere fungal assemblages were similar among sites despite being largely geographically distant. Many organisms comprising the foliar fungal community on F. mandshurica in Far East Russia have similarity to those reported inhabiting F. excelsior in Europe based on previous studies. However, Mycosphaerella sp., the most dominant species in this study and detected in nearly all samples, was associated only with F. mandshurica. Genetic diversity of H. fraxineus was significantly higher in the Far East Russian population than in Europe. In contrast to its aggressive behaviour on Fraxinus excelsior in Europe, H. fraxineus appears to be a benign associate of indigenous F. mandshurica that initially induces quiescent and asymptomatic infections in healthy trees prior to active host colonization normally associated with modification of host tissue during senescence.

Detection of Quiescent Infections with Multiple Elephant Endotheliotropic Herpesviruses (EEHVs), Including EEHV2, EEHV3, EEHV6, and EEHV7, within Lymphoid Lung Nodules or Lung and Spleen Tissue Samples from Five Asymptomatic Adult African Elephants.

More than 80 cases of lethal hemorrhagic disease associated with elephant endotheliotropic herpesviruses (EEHVs) have been identified in young Asian elephants worldwide. Diagnostic PCR tests detected six types of EEHV in blood of elephants with acute disease, although EEHV1A is the predominant pathogenic type. Previously, the presence of herpesvirus virions within benign lung and skin nodules from healthy African elephants led to suggestions that African elephants may be the source of EEHV disease in Asian elephants. Here, we used direct PCR-based DNA sequencing to detect EEHV genomes in necropsy tissue from five healthy adult African elephants. Two large lung nodules collected from culled wild South African elephants contained high levels of either EEHV3 alone or both EEHV2 and EEHV3. Similarly, a euthanized U.S. elephant proved to harbor multiple EEHV types distributed nonuniformly across four small lung nodules, including high levels of EEHV6, lower levels of EEHV3 and EEHV2, and a new GC-rich branch type, EEHV7. Several of the same EEHV types were also detected in random lung and spleen samples from two other elephants. Sanger PCR DNA sequence data comprising 100 kb were obtained from a total of 15 different strains identified, with (except for a few hypervariable genes) the EEHV2, EEHV3, and EEHV6 strains all being closely related to known genotypes from cases of acute disease, whereas the seven loci (4.0 kb) obtained from EEHV7 averaged 18% divergence from their nearest relative, EEHV3. Overall, we conclude that these four EEHV species, but probably not EEHV1, occur commonly as quiescent infections in African elephants. Acute hemorrhagic disease characterized by high-level viremia due to infection by members of the Proboscivirus genus threatens the future breeding success of endangered Asian elephants worldwide. Although the genomes of six EEHV types from acute cases have been partially or fully characterized, lethal disease predominantly involves a variety of strains of EEHV1, whose natural host has been unclear. Here, we carried out genotype analyses by partial PCR sequencing of necropsy tissue from five asymptomatic African elephants and identified multiple simultaneous infections by several different EEHV types, including high concentrations in lymphoid lung nodules. Overall, the results provide strong evidence that EEHV2, EEHV3, EEHV6, and EEHV7 represent natural ubiquitous infections in African elephants, whereas Asian elephants harbor EEHV1A, EEHV1B, EEHV4, and EEHV5. Although a single case of fatal cross-species infection by EEHV3 is known, the results do not support the previous concept that highly pathogenic EEHV1A crossed from African to Asian elephants in zoos.

Immunoassay‐based Techniques for Early and Specific Detection of Latent Postharvest Anthracnose in Mango

AbstractThe postharvest anthracnose pathogen Colletotrichum gloeosporioides inciting latent or quiescent infection of mango was detected in early stages using immunoassay methods. Twenty‐five pathotypes isolated from different agroclimatic zones of Tamil Nadu, Karnataka and Pondicherry, India, revealed the variation in protein profile analysis (SDS‐PAGE). The polyclonal antibodies (PCA) were raised against the unfractioned mycelial protein (UMP) and a 40‐kDa polypeptide present in all pathotypes. Standardization of antigen and antiserum dilutions revealed that an antigen dilution of 1 : 200 (protein concentration of 20 μg/ml) and antiserum dilution of 1 : 100 (protein concentration of 40 μg/ml raised against UMP) and 1 : 200 (protein concentration of 20 μg/ml raised against 40 kDa polypeptide) was found to be optimum for the detection of anthracnose pathogen. Both antisera detected the C. gloeosporioides antigen in enzyme‐linked immunosorbent assays (ELISAs), dot immunobinding assays (DIBAs) and Western blots. The specificity in reaction was compared by isolating other Colletotrichum spp. from various hosts viz., C. lindemuthianum (beans), C. falcatum (sugarcane), C. musae (banana), C. capsici (chillies) and Botryodiplodia theobromae (mango). The antisera generated against UMP revealed the cross‐reaction with other host isolates and mango stem end rot pathogen (B. theobromae). The PCA raised against 40‐kDa polypeptide exhibited the specific reaction with C. gloeosporioides isolates in all the immunoassay techniques. By utilizing both PCA, the presence of latent infection was observed in healthy‐looking leaves, flowers and fruits in orchard conditions. The fruit tissues recorded high absorbance values followed by flowers and leaves in all the detection methods. The ELISA technique was also useful in assessing the pathogen inoculum at various biocontrol formulations sprayed mango trees under field conditions. The fluorescent pseudomonad strains mixture (KFP1 + FP7) amended with chitin sprayed at 30‐day intervals revealed the significant reduction in pathogen load than other formulations and unsprayed control.

Dynamic Response of IFI16 and Promyelocytic Leukemia Nuclear Body Components to Herpes Simplex Virus 1 Infection.

Intrinsic immunity is an aspect of antiviral defense that operates through diverse mechanisms at the intracellular level through a wide range of constitutively expressed cellular proteins. In the case of herpesviruses, intrinsic resistance involves the repression of viral gene expression during the very early stages of infection, a process that is normally overcome by viral tegument and/or immediate-early proteins. Thus, the balance between cellular repressors and virus-counteracting proteins determines whether or not a cell becomes productively infected. One aspect of intrinsic resistance to herpes simplex virus 1 (HSV-1) is conferred by components of promyelocytic leukemia nuclear bodies (PML NBs), which respond to infection by accumulating at sites that are closely associated with the incoming parental HSV-1 genomes. Other cellular proteins, including IFI16, which has been implicated in sensing pathogen DNA and initiating signaling pathways that lead to an interferon response, also respond to viral genomes in this manner. Here, studies of the dynamics of the response of PML NB components and IFI16 to invading HSV-1 genomes demonstrated that this response is extremely rapid, occurring within the first hour after addition of the virus, and that human Daxx (hDaxx) and IFI16 respond more rapidly than PML. In the absence of HSV-1 regulatory protein ICP0, which counteracts the recruitment process, the newly formed, viral-genome-induced PML NB-like foci can fuse with existing PML NBs. These data are consistent with a model involving viral genome sequestration into such structures, thereby contributing to the low probability of initiation of lytic infection in the absence of ICP0. Herpesviruses have intimate interactions with their hosts, with infection leading either to the productive lytic cycle or to a quiescent infection in which viral gene expression is suppressed while the viral genome is maintained in the host cell nucleus. Whether a cell becomes lytically or quiescently infected can be determined through the competing activities of cellular repressors and viral activators, some of which counteract cell-mediated repression. Therefore, the events that occur within the earliest stages of infection can be of crucial importance. This paper describes the extremely rapid response to herpes simplex virus 1 infection of cellular protein IFI16, a sensor of pathogen DNA, and also of the PML nuclear body proteins PML and hDaxx, as revealed by live-cell microscopy. The data imply that these proteins can accumulate on or close to the viral genomes in a sequential manner which may lead to their sequestration and repression.

Clostridium Sacroiliitis (Gas Gangrene) Following Sacroiliac Joint Injection – Case Report and Review of the Literature

An 80-year-old woman presented with chronic lumbosacral pain since her laminectomy and instrumentation 10 years ago. Examination was consistent with left sacroiliitis, and the patient underwent an elective left sacroiliac joint injection. Two days following her procedure she fell and landed on her left hip and on the next day, she presented to the emergency room with acutely worsening left gluteal pain. On evaluation in the emergency department, she was found to be suffering from a fever, headache, nausea, vomiting, and dysuria. A computed tomography (CT) scan was performed and this showed extensive foci of gas throughout the posterior aspect of the left iliopsoas muscle, sacrum, and ileum surrounding the left sacroiliac joint. The patient underwent emergent surgical debridement. The microbiology report of blood culture revealed clostridium perfringens, while her pathology showed necrosis and acute inflammation of fibroadipose tissue, skeletal muscle, and fat. The patient’s hospital course resulted in multi-organ failure and the family elected for comfort care measures only. Unfortunately, she passed away 36 hours later. Septic arthritis is a potential catastrophic complication following intra-articular steroid therapy. The cause of the septic joint can be multifactorial but is likely caused by one of the following processes: direct inoculation of bacteria by the injection, hematogenous seeding of the percutaneous injection tract, or due to activation of a quiescent infection by the injected steroid. Clostridial spores are very resistant to standard aseptic skin preparations, including chlorhexidine and betadine solutions. The only effective methods to eliminate the spores is to heat them at a temperature greater than 100 degrees Celsius for 10 minutes or with use of a 10% potassium hydroxide (KOH) solution. We hypothesize that clostridium spores were present on the patient’s skin from previous stool soiling, and that these were introduced directly into the soft tissue by needle trauma. Rare complications such as this one are scarcely reported in the literature and thus it becomes difficult to adequately identify risk factors or to formulate strategies to improve practice management. Key words: Lower back pain, sacroiliitis, sacroiliac joint injection, clostridium perfringens, gas gangrene

An in vitro model of latency and reactivation of varicella zoster virus in human stem cell-derived neurons.

Varicella zoster virus (VZV) latency in sensory and autonomic neurons has remained enigmatic and difficult to study, and experimental reactivation has not yet been achieved. We have previously shown that human embryonic stem cell (hESC)-derived neurons are permissive to a productive and spreading VZV infection. We now demonstrate that hESC-derived neurons can also host a persistent non-productive infection lasting for weeks which can subsequently be reactivated by multiple experimental stimuli. Quiescent infections were established by exposing neurons to low titer cell-free VZV either by using acyclovir or by infection of axons in compartmented microfluidic chambers without acyclovir. VZV DNA and low levels of viral transcription were detectable by qPCR for up to seven weeks. Quiescently-infected human neuronal cultures were induced to undergo renewed viral gene and protein expression by growth factor removal or by inhibition of PI3-Kinase activity. Strikingly, incubation of cultures induced to reactivate at a lower temperature (34°C) resulted in enhanced VZV reactivation, resulting in spreading, productive infections. Comparison of VZV genome transcription in quiescently-infected to productively-infected neurons using RNASeq revealed preferential transcription from specific genome regions, especially the duplicated regions. These experiments establish a powerful new system for modeling the VZV latent state, and reveal a potential role for temperature in VZV reactivation and disease.

An investigation of herpes simplex virus type 1 latency in a novel mouse dorsal root ganglion model suggests a role for ICP34.5 in reactivation

After a primary lytic infection at the epithelia, herpes simplex virus type 1 (HSV-1) enters the innervating sensory neurons and translocates to the nucleus, where it establishes a quiescent latent infection. Periodically, the virus can reactivate and the progeny viruses spread back to the epithelium. Here, we introduce an embryonic mouse dorsal root ganglion (DRG) culture system, which can be used to study the mechanisms that control the establishment, maintenance and reactivation from latency. Use of acyclovir is not necessary in our model. We examined different phases of the HSV-1 life cycle in DRG neurons, and showed that WT HSV-1 could establish both lytic and latent form of infection in the cells. After reactivating stimulus, the WT viruses showed all markers of true reactivation. In addition, we showed that deletion of the γ(1)34.5 gene rendered the virus incapable of reactivation, even though the virus was clearly able to replicate and persist in a quiescent form in the DRG neurons.

Initial Inoculum and Spatial Dispersal of Colletotrichum gloeosporioides, the Causal Agent of Strawberry Anthracnose Crown Rot.

Anthracnose crown rot (ACR), caused by Colletotrichum gloeosporioides, is a serious disease of strawberry (Fragaria × ananassa) in nurseries and fruiting fields in the southeastern United States. This study was conducted to determine the potential of alternative hosts for initial inoculum source and spread that causes ACR in strawberry nurseries. Results indicated that Parthenocissus quinquefolia is a noncultivated host of C. gloeosporioides in North Carolina and may serve as an initial inoculum source for planting material. Sources of inoculum data were complemented with a 2-year study of disease incidence and spread in simulated nursery production experiments. Sixty days after inoculation of the mother plants in the nursery, three different inoculation levels showed a significant positive correlation (r = 0.78, P < 0.004) with the quiescent infection (QI) incidence on the runner or daughter plants at the end of the nursery production cycle. Runner plant counts from different proportion of mother plants' inoculation treatments indicated that runner plant production was negatively and significantly (P < 0.001) affected by C. gloeosporioides. Infected tips used to produce transplants destined for fruit production resulted in 29.3 and 16.8% mortality in plug trays in 2007 and 2008, respectively. Tracking foliar QI incidence that resulted from dispersal of inoculum from an introduced point source in the nursery showed a sharp decline at 1 m and beyond from the inoculation focus. Although the exponential model (R2 = 0.92 to 0.94) had slightly higher coefficients of determination than the modified power law (R2 = 0.89 to 0.90), residual plots indicated that the modified power law model fit the disease gradient data better than the exponential model in both years. Results from our dispersal study indicated that rogueing of infected plants within a 4-m radius of infection foci would reduce the risk of transferring infected runner plants from the nursery to the fruiting field.

THE ROLE OF EPIDEMIOLOGY DATA IN DEVELOPING INTEGRATED MANAGEMENT OF ANTHRACNOSE IN OLIVES - A REVIEW

Colletotrichum species have the capacity to produce asymptomatic latent (quiescent) infections on various olive tree tissues: during this symptomless biotrophic phase, the pathogen invades the host without killing them and feeds on living cells. Re-infection of the tree by the repeating conidial stage is then responsible for increased anthracnose symptoms during summer, leading to new growths. Normally developed fruit and fruit damaged by abiotic factors, leaves and stems may have asymptomatic infections of Colletotrichum acutatum and C. gloeosporioides in susceptible cultivars ‘Barnea’, ‘Manzanillo’ and ‘Kalamata’. Anthracnose under favourable conditions can also infect less susceptible cultivars. The infection can persist from season to season, depending on olive cultivar, environment, crop management, and pathogen virulence. Brown lesions with anthracnose fungal spores were observed in young green stems and leaves and dieback of young shoot tips of olive susceptible cultivars in orchards in early spring and summer. Flowering, fruit set and immature fruits can be observed during fruit development on a single peduncle; both flowers and fruit set late in the season, in summer rather than spring, and carry fungal infection. Immature rotten fruits are mummified. Colletotrichum fungi overwinter in mummified fruits on the tree, woody tissue and leaves; the fungus has long saprophytic survival ability on dead peduncle and pedicels. Fungal inoculum present year-round throughout the canopy. Hemibiotrophic anthracnose fungal pathogens grow first on living tissue and then cause host death in later, necrotrophic growth. The ability to survive and multiply in the absence of symptoms may explain why anthracnose fungi often cause unexpected crop losses in olives. Successful management of anthracnose relies on understanding the conditions that promote disease development. The complexities of anthracnose epidemiology, including the presence of different species, point to the need for continued research around disease management, particularly regarding new control strategies for Colletotrichum in olives.

Thyroid hormone-dependent epigenetic suppression of herpes simplex virus-1 gene expression and viral replication in differentiated neuroendocrine cells

A global HSV-1 gene repression occurs during latency in sensory neurons where most viral gene transcriptions are suppressed. The molecular mechanisms of gene silencing and how stress factors trigger the reactivation are not well understood. Thyroid hormones are known to be altered due to stress, and with its nuclear receptor impart transcriptional repression or activation depending upon the hormone level. Therefore we hypothesized that triiodothyronine (T3) treatment of infected differentiated neuron like cells would reduce the ability of HSV-1 to produce viral progeny compared to untreated infected cells. Previously we identified putative thyroid hormone receptor elements (TREs) within the promoter regions of HSV-1 thymidine kinase (TK) and other key genes. Searching for a human cell line that can model neuronal HSV-1 infection, we performed HSV-1 infection experiments on differentiated human neuroendocrine cells, LNCaP. Upon androgen deprivation these cells undergo complete differentiation and exhibit neuronal-like morphology and physiology. These cells were readily infected by our HSV-1 recombinant virus, expressing GFP and maintaining many processes iconic of dendritic morphology. Our results demonstrated that differentiated LNCaP cells produced suppressive effects on HSV-1 gene expression and replication compared to its undifferentiated counterpart and T3 treatment has further decreased the viral plaque counts compared to untreated cells. Upon washout of the T3 viral plaque counts were restored, indicating an increase of viral replication. The qRT-PCR experiments using primers for TK showed reduced expression under T3 treatment. ChIP assays using a panel of antibodies for H3 lysine 9 epigenetic marks showed increased repressive marks on the promoter regions of TK. In conclusion we have demonstrated a T3 mediated quiescent infection in differentiated LNCaP cells that has potential to mimic latent infection. In this HSV-1 infection model thyroid hormone treatment caused decreased viral replication, repressed TK expression and increased repressive histone tail marks on the TK promoter.

Transcription of the Herpes Simplex Virus 1 Genome during Productive and Quiescent Infection of Neuronal and Nonneuronal Cells

Herpes simplex virus 1 (HSV-1) can undergo a productive infection in nonneuronal and neuronal cells such that the genes of the virus are transcribed in an ordered cascade. HSV-1 can also establish a more quiescent or latent infection in peripheral neurons, where gene expression is substantially reduced relative to that in productive infection. HSV mutants defective in multiple immediate early (IE) gene functions are highly defective for later gene expression and model some aspects of latency in vivo. We compared the expression of wild-type (wt) virus and IE gene mutants in nonneuronal cells (MRC5) and adult murine trigeminal ganglion (TG) neurons using the Illumina platform for cDNA sequencing (RNA-seq). RNA-seq analysis of wild-type virus revealed that expression of the genome mostly followed the previously established kinetics, validating the method, while highlighting variations in gene expression within individual kinetic classes. The accumulation of immediate early transcripts differed between MRC5 cells and neurons, with a greater abundance in neurons. Analysis of a mutant defective in all five IE genes (d109) showed dysregulated genome-wide low-level transcription that was more highly attenuated in MRC5 cells than in TG neurons. Furthermore, a subset of genes in d109 was more abundantly expressed over time in neurons. While the majority of the viral genome became relatively quiescent, the latency-associated transcript was specifically upregulated. Unexpectedly, other genes within repeat regions of the genome, as well as the unique genes just adjacent the repeat regions, also remained relatively active in neurons. The relative permissiveness of TG neurons to viral gene expression near the joint region is likely significant during the establishment and reactivation of latency. During productive infection, the genes of HSV-1 are transcribed in an ordered cascade. HSV can also establish a more quiescent or latent infection in peripheral neurons. HSV mutants defective in multiple immediate early (IE) genes establish a quiescent infection that models aspects of latency in vivo. We simultaneously quantified the expression of all the HSV genes in nonneuronal and neuronal cells by RNA-seq analysis. The results for productive infection shed further light on the nature of genes and promoters of different kinetic classes. In quiescent infection, there was greater transcription across the genome in neurons than in nonneuronal cells. In particular, the transcription of the latency-associated transcript (LAT), IE genes, and genes in the unique regions adjacent to the repeats persisted in neurons. The relative activity of this region of the genome in the absence of viral activators suggests a more dynamic state for quiescent genomes persisting in neurons.

Comprehensive Analysis of Human Cytomegalovirus MicroRNA Expression during Lytic and Quiescent Infection

BackgroundHuman cytomegalovirus (HCMV) encodes microRNAs (miRNAs) that function as post-transcriptional regulators of gene expression during lytic infection in permissive cells. Some miRNAs have been shown to suppress virus replication, which could help HCMV to establish or maintain latent infection. However, HCMV miRNA expression has not been comprehensively examined and compared using cell culture systems representing permissive (lytic) and semi-permissive vs. non-permissive (latent-like) infection.MethodsViral miRNAs levels and expression kinetics during HCMV infection were determined by miRNA-specific stem-loop RT-PCR. HCMV infected THP-1 (non-permissive), differentiated THP-1 (d-THP-1, semi-permissive) and human embryo lung fibroblasts (HELs, fully-permissive) were examined. The impact of selected miRNAs on HCMV infection (gene expression, genome replication and virus release) was determined by Western blotting, RT-PCR, qPCR, and plaque assay.ResultsAbundant expression of 15 HCMV miRNAs was observed during lytic infection in HELs; highest peak inductions (11- to 1502-fold) occurred at 48 hpi. In d-THP-1s, fourteen mRNAs were detected with moderate induction (3- to 288-fold), but kinetics of expression was generally delayed for 24 h relative to HELs. In contrast, only three miRNAs were induced to low levels (3- to 4-fold) during quiescent infection in THP-1s. Interestingly, miR-UL70-3p was poorly induced in HEL (1.5-fold), moderately in THP-1s (4-fold), and strongly (58-fold) in d-THP-1s, suggesting a potentially specific role for miR-UL70-3p in THP-1s and d-THP-1s. MiR-US33, -UL22A and -UL70 were further evaluated for their impact on HCMV replication in HELs. Ectopic expression of miR-UL22A and miR-UL70 did not affect HCMV replication in HELs, whereas miR-US33 inhibited HCMV replication and reduced levels of HCMV US29 mRNA, confirming that US29 is a target of miR-US33.ConclusionsViral miRNA expression kinetics differs between permissive, semi-permissive and quiescent infections, and miR-US33 down-regulates HCMV replication. These results suggest that miR-US33 may function to impair entry into lytic replication and hence promote establishment of latency.

Identification and characterization of Colletotrichum fructicola causing black spots on young fruits related to bitter rot of pear (Pyrus bretschneideri Rehd.) in China

In recent years, Pyrus bretschneideri Rehd. var. ‘Suli’ has been damaged by a disease characterized by the presence of black spots on young fruit in China, which was always followed by severe bitter rot on matured fruits. The etiology of these symptoms and their relationship with the bitter rot was unknown. A colony was routinely isolated from young and matured ‘Suli’ pear fruits showing black spots and rot symptoms, respectively. This fungal colony was identified as Colletotrichum fructicola based on morphological characteristics and DNA sequence data of 7 regions. C. fructicola was confirmed to be capable of eliciting both black spots and bitter rot symptoms by completion of Koch's Postulates. The results suggested that the disease characterized by the presence of black spots on young fruits is indeed an early stage of the bitter rot observed on matured fruits of ‘Suli’ pear. Variety resistance analysis indicated an obvious resistance variation possessed by the pear varieties generally cultured in China, with black spots only occurring on some pear varieties. Our results also indicated that wounding can break the quiescent infections, enhance the infectivity of C. fructicola, and lead to more rapid rot of young and matured fruits. These results provided a clear clue that C. fructicola can invade some varieties of P. bretschneideri Rehd. directly without entry via wounds, but subsequently turned into quiescent infection and causing black spots on the fruit surface. The quiescent infection acts as a pathogenic factor responsible for bitter rot of matured fruits. The influence of temperature and pH conditions for growth and colony morphology of C. fructicola were also evaluated. The characterization of C. fructicola causing bitter rot of pear (P. bretschneideri) is expected to provide useful information for controlling this economically important disease.

Expression of Herpes Simplex Virus 1 MicroRNAs in Cell Culture Models of Quiescent and Latent Infection

To facilitate studies of herpes simplex virus 1 latency, cell culture models of quiescent or latent infection have been developed. Using deep sequencing, we analyzed the expression of viral microRNAs (miRNAs) in two models employing human fibroblasts and one using rat neurons. In all cases, the expression patterns differed from that in productively infected cells, with the rat neuron pattern most closely resembling that found in latently infected human or mouse ganglia in vivo.

Toxoplasma encephalitis in HIV/AIDS patients admitted to the Douala general hospital between 2004 and 2009: a cross sectional study

BackgroundIt is estimated that about a third of the world’s population is chronically infected with Toxoplasma gondii. Toxoplasma encephalitis (TE), which occurs as a reactivation of quiescent chronic infection, is one of the leading causes of central nervous system (CNS) infection in AIDS. Its diagnosis in most centres still remains difficult. We opted to describe the clinical and radiological features of TE as well as in-hospital outcome and its associated factors.MethodsWe carried out a cross sectional study on the clinical case notes of adult patients admitted and treated for TE at the Douala General Hospital, Cameroon between January 1st 2004 to December 31st 2009.ResultsOf 672 patients admitted during the study period, 14.4% (97/672) had TE. The mean age was 36.9 ± 14.1 years and the median CD4 cell count was 68/mm3 (IQR): 43 – 103). Headache and fever were the most common presenting symptoms in 92.8% (90/97) and 87.6% (85/97) of patients. Annular contrast enhanced lesions were the most common brain scan finding in 81.4% (79/97) of patients. In-hospital mortality was 29.9% (29/97). Altered sensorium, presence of focal signs, neck stiffness and low CD4 cell count were factors associated with mortality. Adjusting for low CD4 count, altered sensorium remained strongly associated with fatality, adjusted odd ratio (AOR) 3.5 (95% CI 1.2 – 10.5).ConclusionToxoplasma encephalitis is common among AIDS patients in Douala. Its high case fatality warrants adequate and compliant prophylactic therapy in severely immune depressed patients as well as early initiation of antiretroviral therapy in HIV-infected patients.

Virus Infections in the Nervous System

Virus infections usually begin in peripheral tissues and can invade the mammalian nervous system (NS), spreading into the peripheral (PNS) and more rarely the central (CNS) nervous systems. The CNS is protected from most virus infections by effective immune responses and multilayer barriers. However, some viruses enter the NS with high efficiency via the bloodstream or by directly infecting nerves that innervate peripheral tissues, resulting in debilitating direct and immune-mediated pathology. Most viruses in the NS are opportunistic or accidental pathogens, but a few, most notably the alpha herpesviruses and rabies virus, have evolved to enter the NS efficiently and exploit neuronal cell biology. Remarkably, the alpha herpesviruses can establish quiescent infections in the PNS, with rare but often fatal CNS pathology. Here we review how viruses gain access to and spread in the well-protected CNS, with particular emphasis on alpha herpesviruses, which establish and maintain persistent NS infections.