Tobacco Necrosis Virus D (TNV-D), in the genus Betanecrovirus (family Tombusviridae), possesses a single-stranded, positive-sense RNA genome containing six open reading frames (ORFs). Two 5'-proximal ORFs (1 and 2) encode overlapping polypeptides of 22 and 82 kDa (p22 and p82, respectively) which are both required for replication. The p22 auxiliary protein contains no replication motifs but the C-terminal region, downstream of a leaky stop codon, encodes a 60 kDa polypeptide (p60) which contains conserved RNA-dependent RNA polymerase (RdRP) motifs. Here we have expressed and purified recombinant p60 and show that in vitro it binds and efficiently synthesises both TNV-D RNA and Satellite tobacco necrosis virus C RNA. Alanine scanning mutagenesis of conserved amino acids in characteristic motifs in p60 revealed that some mutations significantly reduced RNA synthesis but mutating the second asparagine residue in the conserved GDD box was lethal. The effects of mutating identical amino acids in p60 on virus replication in vivo were examined in Nicotiana benthamiana plants following infection with RNA transcribed from wild type (wt) and mutant constructs. In inoculated leaves the behaviour of the mutants paralleled the in vitro data but systemic infection was precluded in all but one mutant which had reverted to wt. This study is the first to demonstrate the nucleic acid-binding and synthetic capabilities of a betanecrovirus polymerase.
Few comprehensive studies have searched for viruses in infants and young children with community-acquired pneumonia (CAP) in China. The aim of this study was to investigate the roles of human herpes viruses (HHVs) and other respiratory viruses in CAP not caused by typical bacterial infection and to determine their prevalence and clinical significance.
Induced sputum (IS) samples were collected from 354 hospitalised patients (infants, n = 205; children, n = 149) with respiratory illness (CAP or non-CAP) admitted to Wenling Hospital of China. We tested for HHVs and respiratory viruses using PCR-based assays. The epidemiological profiles were also analysed.
High rate of virus detection (more than 98%) and co-infection (more than 80%) were found among IS samples from 354 hospitalised infants and children with respiratory illness in this study. Of 273 CAP samples tested, CMV (91.6%), HHV-6 (50.9%), RSV (37.4%), EBV (35.5%), HBoV (28.2%), HHV-7 (18.3%) and rhinovirus (17.2%) were the most commonly detected viruses. Of 81 non- CAP samples tested, CMV (63%), RSV (49.4%), HHV-6 (42%), EBV (24.7%), HHV-7 (13.6%) and HBoV (8.6%) were the dominant viruses detected. The prevalence of several viral agents (rhinovirus, bocavirus, adenovirus and CMV) among IS samples of CAP were significantly higher than that of non-CAP control group. We also found the prevalence of RSV coinfection with HHVs was also higher among CAP group than that of non-CAP control.
With sensitive molecular detection techniques and IS samples, high rates of viral identification were achieved in infants and young children with respiratory illness in a rural area of China. The clinical significance of rhinovirus, bocavirus, adenovirus and HHV (especially CMV) infections should receive greater attention in future treatment and prevention studies of CAP in infants and children.
Human parechoviruses are known to cause asymptomatic to severe clinical illness predominantly respiratory and gastroenetric infections. Despite their global prevalence, epidemiological studies have not been performed in Pakistan. In this study, we retrospectively analyzed 110 fecal specimen and found 26 (24%) positive for viral RNA with HPeV-10 (n = 3, 23%), HPeV-13 (n = 4, 31%) and HPeV-15 (n = 6, 46%) genotypes. Clinical features of patients with different HPeV genotypes were compared. All HPeV positive children were aged ≤4 years (mean 13.92 months). The male-to-female ratio was 1: 1.17 (46.2 vs 53.8%) with significant association (p = .031) to HPeV infectivity. HPeV-10 and -13 were found during summer while HPeV-15 was only detected during late winter season. Disease symptoms were more severe in children infected with HPeV-10 and -13 as compared to HPeV-15. Fever and vomiting were observed in 100% cases of HPeV-10 and -13 while only 17% patients of HPeV-15 had these complaints. Phylogenetic analyses showed that HPeV-10, -13 and -15 strains found in this study have 9–13%, 16.8% and 21.8% nucleotide divergence respectively from the prototype strains and were clustered to distinct genetic lineages. This is the first report of HPeV-15 infection in humans although first identified in rhesus macaques. The arginine-glycine-aspartic acid (RGD) motif present at the C-terminal of VP1 responsible for the viral attachment to cellular integrins was not found in all of these strains. In conclusion, these findings enhance our knowledge related to the epidemiology and genetic diversity of the HPeV in Pakistan and support the need for continued laboratory based surveillance programs especially in infants and neonatal clinical settings. Further, the parechovirus pathogenesis, cross-species transmission and disease reservoirs must be ascertained to adopt better prevention measures.
Using random PCR in combination with next-generation sequencing, a novel parvovirus was detected in the brain of a young harbor seal (Phoca vitulina) with chronic non-suppurative meningo-encephalitis that was rehabilitated at the Seal Rehabilitation and Research Centre (SRRC) in the Netherlands. In addition, two novel viruses belonging to the family Anelloviridae were detected in the lungs of this animal. Phylogenetic analysis of the coding sequence of the novel parvovirus, tentatively called Seal parvovirus, indicated that this virus belonged to the genus Erythrovirus, to which human parvovirus B19 also belongs. Although no other seals with similar signs were rehabilitated in SRRC in recent years, a prevalence study of tissues of seals from the same area collected in the period 2008-2012 indicated that the Seal parvovirus has circulated in the harbor seal population at least since 2008. The presence of the Seal parvovirus in the brain was confirmed by real-time PCR and in vitro replication. Using in situ hybridization, we showed for the first time that a parvovirus of the genus Erythrovirus was present in the Virchow-Robin space and in cerebral parenchyma adjacent to the meninges. These findings showed that a parvovirus of the genus Erythrovirus can be involved in central nervous system infection and inflammation, as has also been suspected but not proven for human parvovirus B19 infection.
Viruses of the family Polyomaviridae infect a wide variety of avian and mammalian hosts with a broad spectrum of outcomes including asymptomatic infection, acute systemic disease, and tumor induction. In this study a novel polyomavirus, the African elephant polyomavirus 1 (AelPyV-1) found in a protruding hyperplastic fibrous lesion on the trunk of an African elephant (Loxodonta africana) was characterized. The AelPyV-1 genome is 5722 bp in size and is one of the largest polyomaviruses characterized to date. Analysis of the AelPyV-1 genome reveals five putative open-reading frames coding for the classic small and large T antigens in the early region, and the VP1, VP2 and VP3 capsid proteins in the late region. In the area preceding the VP2 start codon three putative open-reading frames, possibly coding for an agnoprotein, could be localized. A regulatory, non-coding region separates the 2 coding regions. Unique for polyomaviruses is the presence of a second 854 bp long non-coding region between the end of the early region and the end of the late region. Based on maximum likelihood phylogenetic analyses of the large T antigen of the AelPyV-1 and 61 other polyomavirus sequences, AelPyV-1 clusters within a heterogeneous group of polyomaviruses that have been isolated from bats, new world primates and rodents.
Activation of a host DNA damage response (DDR) is essential for DNA replication of minute virus of canines (MVC), a member of the genus Bocavirus of the Parvoviridae family; however, the mechanism by which DDR contributes to viral DNA replication is unknown. In the current study, we demonstrate that MVC infection triggers the intra-S-phase arrest to slow down host cellular DNA replication and to recruit cellular DNA replication factors for viral DNA replication. The intra-S-phase arrest is regulated by ATM (ataxia telangiectasia-mutated kinase) signaling in a p53-independent manner. Moreover, we demonstrate that SMC1 (structural maintenance of chromosomes 1) is the key regulator of the intra-S-phase arrest induced during infection. Either knockdown of SMC1 or complementation with a dominant negative SMC1 mutant blocks both the intra-S-phase arrest and viral DNA replication. Finally, we show that the intra-S-phase arrest induced during MVC infection was caused neither by damaged host cellular DNA nor by viral proteins but by replicating viral genomes physically associated with the DNA damage sensor, the Mre11-Rad50-Nbs1 (MRN) complex. In conclusion, the feedback loop between MVC DNA replication and the intra-S-phase arrest is mediated by ATM-SMC1 signaling and plays a critical role in MVC DNA replication. Thus, our findings unravel the mechanism underlying DDR signaling-facilitated MVC DNA replication and demonstrate a novel strategy of DNA virus-host interaction.
Human bocavirus 1 (HBoV1) is an emerging human-pathogenic respiratory virus. We characterized two important features of HBoV1 infection in polarized primary human airway epithelia (HAE). Apical HBoV1 infection of HAE at a low multiplicity of infection causes disruption of the tight junction barrier, loss of cilia, and epithelial cell hypertrophy, which are hallmarks of the airway epithelial damage caused by HBoV1 infection. HBoV1 also infects HAE from the basolateral surface productively, although less efficiently, and this also leads to the characteristic airway epithelial damage.
Recombinant Adeno-associated virus vectors (rAAV) are widely used for gene delivery and multiple naturally occurring serotypes have been harnessed to target cells in different tissues and organs including the brain. Here, we provide a detailed and quantitative analysis of the transduction profiles of rAAV vectors based on six of the most commonly used serotypes (AAV1, AAV2, AAV5, AAV6, AAV8, AAV9) that allows systematic comparison and selection of the optimal vector for a specific application. In our studies we observed marked differences among serotypes in the efficiency to transduce three different brain regions namely the striatum, hippocampus and neocortex of the mouse. Despite the fact that the analyzed serotypes have the general ability to transduce all major cell types in the brain (neurons, microglia, astrocytes and oligodendrocytes), the expression level of a reporter gene driven from a ubiquitous promoter varies significantly for specific cell type / serotype combinations. For example, rAAV8 is particularly efficient to drive transgene expression in astrocytes while rAAV9 appears well suited for the transduction of cortical neurons. Interestingly, we demonstrate selective retrograde transport of rAAV5 along axons projecting from the ventral part of the entorhinal cortex to the dentate gyrus. Furthermore, we show that self-complementing rAAV can be used to significantly decrease the time required for the onset of transgene expression in the mouse brain.
AAV vectors have shown great promise for clinical gene therapy (GT), but pre-existing human immunity against the AAV capsid often limits transduction. Thus, testing promising AAV-based GT approaches in an animal model with similar pre-existing immunity could better predict clinical outcome. Sheep have long been used for basic biological and preclinical studies. Moreover, we have re-established a line of sheep with severe hemophilia A (HA). Given the impetus to use AAV-based GT to treat hemophilia, we characterized the pre-existing ovine humoral immunity to AAV. ELISA revealed naturally-occurring antibodies to AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9. For AAV2, AAV8, and AAV9 these inhibit transduction in a luciferase-based neutralization assay. Epitope mapping identified peptides that were common to the capsids of all AAV serotypes tested (AAV2, AAV5, AAV8 and AAV9), with each animal harboring antibodies to unique and common capsid epitopes. Mapping using X-ray crystallographic AAV capsid structures demonstrated that these antibodies recognized both surface epitopes and epitopes located within regions of the capsid that are internal or buried in the capsid structure. These results suggest that sheep harbor endogenous AAV, which induces immunity to both intact capsid and to capsid epitopes presented following proteolysis during the course of infection. In conclusion, their clinically relevant physiology and the presence of naturally-occurring antibodies to multiple AAV serotypes collectively make sheep a unique model in which to study GT for HA, and other diseases, and develop strategies to circumvent the clinically important barrier of pre-existing AAV immunity.
Oxidative stress is one of the major factors that trigger photoreceptor apoptosis. To investigate whether resveratrol, a potent antioxidant and small molecule activator of the FoxO pathway, would be neuroprotective against photoreceptor cell death in a rodent model of retinal detachment.
Retinal detachment was created in adult Brown Norway rats by subretinal injection of sodium hyaluronate. The animals were treated daily with vehicle or resveratrol (20mg/kg) intraperitoneal injection. Photoreceptor death was assessed by counting the number of apoptotic cells with TdT-dUTP terminal nick-end labeling (TUNEL) and measurement of the outer nuclear layer (ONL) thickness 3 days after RD. Changes in expression of FoxO1a, FoxO3a, and FoxO4 were analyzed by western blot. The activity of caspase 3, caspase 8, caspase 9, spectrin and their cleavage forms were studied.
Three days after retinal detachment, caspase 3, caspase 8 and caspase 9 were significantly activated in the detached retina. Spectrin cleavage products at 120 and 145 kDa were also detected. Both caspase and calpain activation are involved in apoptotic photoreceptor cell death in detached retinas. Treatment with resveratrol increases FoxO1a, FoxO3a, and FoxO4 protein expression in detached retinas only. Resveratrol treatment decreases activation of intrinsic and extrinsic caspase apoptotic pathways triggered by RD. The number of TUNEL-positive cells decreases from 1301±51 cells/mm2 in control groups to 430±35 cells/mm2 in treatment groups (p<0.05). Resveratrol treatment also demonstrates 59% less ONL thickness loss compared to controls.
Resveratrol treatment up-regulates the FoxO family and blocks Caspase3, 8, and 9 activation. Resveratrol has the potential to be used as a novel therapeutic agent for preventing vision loss in diseases characterized by photoreceptor detachment.
Hantaan virus (HTNV) could cause a severe lethal hemorrhagic fever with renal syndrome (HFRS) in humans. Despite a limited understanding of the pathogenesis of HFRS, the importance of host-related immune responses in the pathogenesis of HFRS has been widely recognized. CD100/Sema4D has been demonstrated to play an important role in physiological and pathological immune responses, but the functional role of CD100 in infectious diseases has only been inadequately reported. The aim of this study was to investigate the pathological significance of CD100 in patients after HTNV infection.
Blood samples were collected from 99 hospitalized patients in Tangdu Hospital and 27 health controls. The level of soluble CD100 (sCD100) in plasma were quantified by ELISA and the relationship between sCD100 and the disease course or severity were analyzed. The expressions of membrane CD100 on various subpopulations of peripheral blood mononuclear cell (PBMC) were analyzed by flow cytometry. The results showed that sCD100 level in acute phase of HFRS was significantly higher in patients than that in healthy controls (P<0.0001) and the sCD100 level declined in convalescent phase. Multivariate model analysis showed that platelet count, white blood cell count, serum creatinine level and blood urea nitrogen level were associated with sCD100 levels and contributed independently to the elevated sCD100 levels. The expression of membrane CD100 on PBMCs decreased in the acute phase of HFRS patients compared with that of the normal controls and recovered in the convalescent phase.
We reported the elevated level of plasma sCD100 in HFRS patients and the elevated level might be a result from the shedding of membrane CD100 on PBMC. The elevated level of sCD100 was associated with disease severity, suggesting that sCD100 might be a cause or a consequence of progression of HFRS. The underlying mechanisms should be explored further.
G-quadruplexes are tetraplex structures of nucleic acids that can form in G-rich sequences. Their presence and functional role have been established in telomeres, oncogene promoters and coding regions of the human chromosome. In particular, they have been proposed to be directly involved in gene regulation at the level of transcription. Because the HIV-1 Nef protein is a fundamental factor for efficient viral replication, infectivity and pathogenesis invitro and invivo, we investigated G-quadruplex formation in the HIV-1 nef gene to assess the potential for viral inhibition through G-quadruplex stabilization. A comprehensive computational analysis of the nef coding region of available strains showed the presence of three conserved sequences that were uniquely clustered. Biophysical testing proved that G-quadruplex conformations were efficiently stabilized or induced by G-quadruplex ligands in all three sequences. Upon incubation with a G-quadruplex ligand, Nef expression was reduced in a reporter gene assay and Nef-dependent enhancement of HIV-1 infectivity was significantly repressed in an antiviral assay. These data constitute the first evidence of the possibility to regulate HIV-1 gene expression and infectivity through G-quadruplex targeting and therefore open a new avenue for viral treatment.
Viral Hemorrhagic Septicemia virus (VHSv) is one of the world's most serious fish pathogens, infecting >80 marine, freshwater, and estuarine fish species from Eurasia and North America. A novel and especially virulent strain – IVb – appeared in the Great Lakes in 2003, has killed many game fish species in a series of outbreaks in subsequent years, and shut down interstate transport of baitfish. Cell culture is the diagnostic method approved by the USDA-APHIS, which takes a month or longer, lacks sensitivity, and does not quantify the amount of virus. We thus present a novel, easy, rapid, and highly sensitive real-time quantitative reverse transcription PCR (qRT-PCR) assay that incorporates synthetic competitive template internal standards for quality control to circumvent false negative results. Results demonstrate high signal-to-analyte response (slope = 1.00±0.02) and a linear dynamic range that spans seven orders of magnitude (R2 = 0.99), ranging from 6 to 6,000,000 molecules. Infected fishes are found to harbor levels of virus that range to 1,200,000 VHSv molecules/106
actb1 molecules with 1,000 being a rough cut-off for clinical signs of disease. This new assay is rapid, inexpensive, and has significantly greater accuracy than other published qRT-PCR tests and traditional cell culture diagnostics.
Porcine epidemic diarrhea virus (PEDV) causes severe economic losses in the swine industry in China and other Asian countries. Infection usually leads to an acute, often lethal diarrhea in piglets. Despite the impact of the disease, no system is yet available to manipulate the viral genome which has severely hampered research on this virus until today. We have established a reverse genetics system for PEDV based on targeted RNA recombination that allows the modification of the 3′-end of the viral genome, which encodes the structural proteins and the ORF3 protein. Using this system, we deleted the ORF3 gene entirely from the viral genome and showed that the ORF3 protein is not essential for replication of the virus in vitro. In addition, we inserted heterologous genes (i.e. the GFP and Renilla luciferase genes) at two positions in the viral genome, either as an extra expression cassette or as a replacement for the ORF3 gene. We demonstrated the expression of both GFP and Renilla luciferase as well as the application of these viruses by establishing a convenient and rapid virus neutralization assay. The new PEDV reverse genetics system will enable functional studies of the structural proteins and the accessory ORF3 protein and will allow the rational design and development of next generation PEDV vaccines.
Conventional non-viral gene transfer uses bacterial plasmid DNA containing antibiotic resistance genes, cis-acting bacterial sequence elements, and prokaryotic methylation patterns that may adversely affect transgene expression and vector stability in vivo. Here, we describe novel replicative forms of a eukaryotic vector DNA that consist solely of an expression cassette flanked by adeno-associated virus (AAV) inverted terminal repeats. Extensive structural analyses revealed that this AAV-derived vector DNA consists of linear, duplex molecules with covalently closed ends (termed closed-ended, linear duplex, or “CELiD”, DNA). CELiD vectors, produced in Sf9 insect cells, require AAV rep gene expression for amplification. Amounts of CELiD DNA produced from insect cell lines stably transfected with an ITR-flanked transgene exceeded 60 mg per 5×109 Sf9 cells, and 1–15 mg from a comparable number of parental Sf9 cells in which the transgene was introduced via recombinant baculovirus infection. In mice, systemically delivered CELiD DNA resulted in long-term, stable transgene expression in the liver. CELiD vectors represent a novel eukaryotic alternative to bacterial plasmid DNA.
Diseases caused by dengue virus (DV) infection vary in severity, with symptoms ranging from mild fever to life threatening dengue hemorrhage fever (DHF) and dengue shock syndrome (DSS). Clinical studies have shown that significant decrease in the level of lipoproteins is correlated with severe illness in DHF/DSS patients. Available evidence also indicates that lipoproteins including high-density lipoprotein (HDL) and low-density lipoprotein (LDL) are able to facilitate cell entry of HCV or other flaviviruses via corresponding lipoprotein receptors. In this study, we found that pre-incubation of DV with human serum leads to an enhanced DV infectivity in various types of cells. Such enhancement could be due to interactions between serum components and DV particles. Through co-immunoprecipitation we revealed that apolipoprotein A-I (ApoA-I), the major protein component in HDL, is associated with DV particles and is able to promote DV infection. Based on that observation, we further found that siRNA knockdown of the scavenger receptor class B type I (SR-BI), the cell receptor of ApoA-I, abolished the activity of ApoA-I in enhancement of DV infection. This suggests that ApoA-I bridges DV particles and cell receptor SR-BI and facilitates entry of DV into cells. FACS analysis of cell surface dengue antigen after virus absorption further confirmed that ApoA-I enhances DV infection via promoting initial attachment of the virus to cells. These findings illustrate a novel entry route of DV into cells, which may provide insights into the functional importance of lipoproteins in dengue pathogenesis.
Our previous studies demonstrated that the cell culture-grown hepatitis C virus of genotype 2a (HCVcc) uses apolipoprotein E (apoE) to mediate its attachment to the surface of human hepatoma Huh-7.5 cells. ApoE mediates HCV attachment by binding to the cell surface heparan sulfate (HS) which is covalently attached to the core proteins of proteoglycans (HSPGs). In the present study, we further determined the physiological importance of apoE and HSPGs in the HCV attachment using a clinical HCV of genotype 1b (HCV1b) obtained from hepatitis C patients and human embryonic stem cell-differentiated hepatocyte-like cells (DHHs). DHHs were found to resemble primary human hepatocytes. Similar to HCVcc, HCV1b was found to attach to the surface of DHHs by the apoE-mediated binding to the cell surface HSPGs. The apoE-specific monoclonal antibody, purified HSPGs, and heparin were all able to efficiently block HCV1b attachment to DHHs. Similarly, the removal of heparan sulfate from cell surface by treatment with heparinase suppressed HCV1b attachment to DHHs. More significantly, HCV1b attachment was potently inhibited by a synthetic peptide derived from the apoE receptor-binding region as well as by an HSPG-binding peptide. Likewise, the HSPG-binding peptide prevented apoE from binding to heparin in a dose-dependent manner, as determined by an in vitro heparin pull-down assay. Collectively, these findings demonstrate that HSPGs serve as major HCV attachment receptors on the surface of human hepatocytes to which the apoE protein ligand on the HCV envelope binds.
We present a detailed characterization of a single-cycle infection of the bocavirus minute virus of canines (MVC) in canine WRD cells. This has allowed identification of an additional smaller NS protein that derives from an mRNA spliced within the NS gene that had not been previously reported. In addition, we have identified a role for the viral NP1 protein during infection. NP1 is required for read-through of the MVC internal polyadenylation site and, thus, access of the capsid gene by MVC mRNAs. Although the mechanism of NP1's action has not yet been fully elucidated, it represents the first parvovirus protein to be implicated directly in viral RNA processing.
Adeno-associated virus (AAV) mediated gene expression is a powerful tool for gene therapy and preclinical studies. A comprehensive analysis of CNS cell type tropism, expression levels and biodistribution of different capsid serotypes has not yet been undertaken in neonatal rodents. Our previous studies show that intracerebroventricular injection with AAV2/1 on neonatal day P0 results in widespread CNS expression but the biodistribution is limited if injected beyond neonatal day P1. To extend these observations we explored the effect of timing of injection on tropism and biodistribution of six commonly used pseudotyped AAVs delivered in the cerebral ventricles of neonatal mice. We demonstrate that AAV2/8 and 2/9 resulted in the most widespread biodistribution in the brain. Most serotypes showed varying biodistribution depending on the day of injection. Injection on neonatal day P0 resulted in mostly neuronal transduction, whereas administration in later periods of development (24–84 hours postnatal) resulted in more non-neuronal transduction. AAV2/5 showed widespread transduction of astrocytes irrespective of the time of injection. None of the serotypes tested showed any microglial transduction. This study demonstrates that both capsid serotype and timing of injection influence the regional and cell-type distribution of AAV in neonatal rodents, and emphasizes the utility of pseudotyped AAV vectors for translational gene therapy paradigms.
Recombinant adeno-associated virus (AAV) vectors are versatile tools for gene transfer to the central nervous system (CNS) and proof-of-concept studies in adult rodents have shown that the use of cell type-specific promoters is sufficient to target AAV-mediated transgene expression to glia. However, neurological disorders caused by glial pathology usually have an early onset. Therefore, modelling and treatment of these conditions require expanding the concept of targeted glial transgene expression by promoter selectivity for gene delivery to the immature CNS. Here, we have investigated the AAV-mediated green fluorescent protein (GFP) expression driven by the myelin basic protein (MBP) or glial fibrillary acidic protein (GFAP) promoters in the developing mouse brain. Generally, the extent of transgene expression after infusion at immature stages was widespread and higher than in adults. The GFAP promoter-driven GFP expression was found to be highly specific for astrocytes following vector infusion to the brain of neonates and adults. In contrast, the selectivity of the MBP promoter for oligodendrocytes was poor following neonatal AAV delivery, but excellent after vector injection at postnatal day 10. To extend these findings obtained in naïve mice to a disease model, we performed P10 infusions of AAV-MBP-GFP in aspartoacylase (ASPA)-deficient mouse mutants presenting with early onset oligodendrocyte pathology. Spread of GFP expression and selectivity for oligodendrocytes in ASPA-mutants was comparable with our observations in normal animals. Our data suggest that direct AAV infusion to the developing postnatal brain, utilising cellular promoters, results in targeted and long-term transgene expression in glia. This approach will be relevant for disease modelling and gene therapy for the treatment of glial pathology.
Replication of RNA viruses in insect cells triggers an antiviral defense that is mediated by RNA interference (RNAi) which generates viral-derived small interfering RNAs (siRNAs). However, it is not known whether an antiviral RNAi response is also induced in insects by reoviruses, whose double-stranded RNA genome replication is thought to occur within core particles. Deep sequencing of small RNAs showed that when the small brown planthopper (Laodelphax striatellus) was infected by Rice black-streaked dwarf virus (RBSDV) (Reoviridae; Fijivirus), more viral-derived siRNAs accumulated than when the vector insect was infected by Rice stripe virus (RSV), a negative single-stranded RNA virus. RBSDV siRNAs were predominantly 21 and 22 nucleotides long and there were almost equal numbers of positive and negative sense. RBSDV siRNAs were frequently generated from hotspots in the 5′- and 3′-terminal regions of viral genome segments but these hotspots were not associated with any predicted RNA secondary structures. Under laboratory condition, L. striatellus can be infected simultaneously with RBSDV and RSV. Double infection enhanced the accumulation of particular genome segments but not viral coat protein of RBSDV and correlated with an increase in the abundance of siRNAs derived from RBSDV. The results of this study suggest that reovirus replication in its insect vector potentially induces an RNAi-mediated antiviral response.
Foot-and-mouth disease virus (FMDV) initiates infection by adhering to integrin receptors on target cells, followed by cell entry and disassembly of the virion through acidification within endosomes. Mild heating of the virions also leads to irreversible dissociation into pentamers, a characteristic linked to reduced vaccine efficacy. In this study, the structural stability of intra- and inter-serotype chimeric SAT2 and SAT3 virus particles to various conditions including low pH, mild temperatures or high ionic strength, was compared. Our results demonstrated that while both the SAT2 and SAT3 infectious capsids displayed different sensitivities in a series of low pH buffers, their stability profiles were comparable at high temperatures or high ionic strength conditions. Recombinant vSAT2 and intra-serotype chimeric viruses were used to map the amino acid differences in the capsid proteins of viruses with disparate low pH stabilities. Four His residues at the inter-pentamer interface were identified that change protonation states at pH 6.0. Of these, the H145 of VP3 appears to be involved in interactions with A141 in VP3 and K63 in VP2, and may be involved in orientating H142 of VP3 for interaction at the inter-pentamer interfaces.
Tuberous sclerosis complex (TSC) is an autosomal dominant disorder due to mutations in either TSC1 or TSC2 that affects many organs with hamartomas and tumors. TSC-associated brain lesions include subependymal nodules, subependymal giant cell astrocytomas and tubers. Neurologic manifestations in TSC comprise a high frequency of mental retardation and developmental disorders including autism, as well as epilepsy. Here, we describe a new mouse model of TSC brain lesions in which complete loss of Tsc1 is achieved in multiple brain cell types in a stochastic pattern. Injection of an adeno-associated virus vector encoding Cre recombinase into the cerebral ventricles of mice homozygous for a Tsc1 conditional allele on the day of birth led to reduced survival, and pathologic findings of enlarged neurons, cortical heterotopias, subependymal nodules, and hydrocephalus. The severity of clinical and pathologic findings as well as survival was shown to be dependent upon the dose and serotype of Cre virus injected. Although several other models of TSC brain disease exist, this model is unique in that the pathology reflects a variety of TSC-associated lesions involving different numbers and types of cells. This model provides a valuable and unique addition for therapeutic assessment.
Eight novel human polyomaviruses have been discovered since 2007. Prevalence rates and tissue tropism for the most recent members HPyV 6, 7, 9, TSPyV and MWPyV are largely unknown. We used real-time PCR to determine the presence of HPyV 6, 7, 9, TSPyV and MWPyV in feces (n = 263), urine (n = 189), blood (n = 161), respiratory swabs (n = 1385) and cerebrospinal fluid (n = 171) from both healthy control children and children and adults undergoing diagnostic testing. Whole genome sequencing was able to be performed on 9 MWPyV positive specimens. Novel polyomaviruses were only detected in respiratory swabs and feces, with no detections of HPyV 9 in any sample type. MWPyV was found to be the most prevalent novel polyomavirus, being detected in 18 (1.5%) respiratory specimens from symptomatic patients, 16 (9.8%) respiratory sample from healthy control children, 11 (5.9%) fecal specimens from patient suffering gastrointestinal illness, and in 13 (15.3%) of feces from healthy control children. MWPyV was found only in respiratory and fecal specimens from children, the oldest being 9 years old. HPyV 6, 7, 9 and TSPyV were also detected in respiratory specimens and fecal specimens at low prevalence (<1.3%). The majority of these detections were found in immunocompromised patients. Our findings suggest that MWPyV can result in a subclinical infection, persistent or intermittent shedding, particularly in young children. The other novel polyomaviruses were also found in respiratory and fecal specimens, but at lower prevalence and most commonly in immunocompromised individuals.
Schmallenberg virus (SBV) is a recently discovered Bunyavirus associated mainly with abortions, stillbirths and malformations of the skeletal and central nervous system (CNS) in newborn ruminants. In this study, a detailed immunophenotyping of the inflammatory cells of the CNS of affected animals was carried out in order to increase our understanding of SBV pathogenesis. A total of 82 SBV-polymerase chain reaction (PCR) positive neonatal ruminants (46 sheep lambs, 34 calves and 2 goat kids) were investigated for the presence of inflammation in the brain and spinal cord. The study focused on 15 out of 82 animals (18.3%) showing inflammation in the CNS. All 15 neonates displayed lymphohistiocytic meningoencephalomyelitis affecting most frequently the mesencephalon and the parietal and temporal lobes. The majority of infiltrating cells were CD3-positive T cells, followed by CD79α-positive B cells and CD68-positive microglia/macrophages. Malformations like por- and hydranencephaly, frequently found in the temporal lobe, showed associated demyelination and axonal loss. SBV antigen was detected in 37 out of 82 (45.1%) neonatal brains by immunohistochemistry. In particular, SBV antigen was found in 93.3% (14 out of 15 ruminants) and 32.8% (22 out of 67 ruminants) of animals with and without encephalitis, respectively. Highest amounts of virus-protein expression levels were found in the temporal lobe. Our findings suggest that: (i) different brain regions display differential susceptibility to SBV infection; (ii) inflammatory cells in the CNS are found only in a minority of virus infected animals; (iii) malformations occur in association with and without inflammation in the CNS; and (iv) viral antigen is strongly associated with the presence of inflammation in naturally infected animals. Further studies are required to explore the cell tropism and pathogenesis of SBV infection in ruminants.