- Volume 98, Issue 7, 2017

The relationship between hepatitis B virus (HBV) infection and lipid accumulation remains largely unknown. In this study, we investigated the effect of HBV propagation on lipid droplet growth in HBV-infected cells and HBV-producing cell lines, HepG2.2.15 and HBV-inducible Hep38.7-Tet. The amount of intracellular triglycerides was significantly reduced in HBV-infected and HBV-producing cells compared with HBV-lacking control cells. Electron and immunofluorescent microscopic analyses showed that the average size of a single lipid droplet (LD) was significantly less in the HBV-infected and HBV-producing cells than in the HBV-lacking control cells. Cell death-inducing DFF45-like effectors (CIDEs) B and C (CIDEB and CIDEC), which are involved in LD expansion for the improvement of lipid storage, were expressed at a significantly lower level in HBV-infected or HBV-producing cells than in HBV-lacking control cells, while CIDEA was not detected in those cells regardless of HBV production. The activity of the CIDEB and CIDEC gene promoters was impaired in HBV-infected or HBV-producing cells compared to HBV-lacking control cells, while CIDEs potentiated HBV core promoter activity. The amount of HNF4α, that can promote the transcription of CIDEB was significantly lower in HBV-producing cells than in HBV-lacking control cells. Knockout of CIDEB or CIDEC significantly reduced the amount of supernatant HBV DNA, intracellular viral RNA and nucleocapsid-associated viral DNA, while the expression of CIDEB or CIDEC recovered HBV production in CIDEB- or CIDEC-knockout cells. These results suggest that HBV regulates its own viral replication via CIDEB and CIDEC.

The seven in absentia homologue 1 (Siah-1) protein is an E3 ubiquitin ligase that induces ubiquitin-dependent proteasomal degradation of HBx, the principal regulatory protein of hepatitis B virus (HBV); however, its role in HBV propagation remains unknown. Here, we found that HBx upregulates Siah-1 levels in HepG2 but not in Hep3B cells, in which p53 is absent. For this effect, HBx sequentially activated ataxia telangiectasia mutated kinase and checkpoint kinase 2 via phosphorylation at the Ser-1981 and Thr-68 residues, respectively, which led to the activation of p53 via phosphorylation at the Ser-15 and Ser-20 residues. As a result, HBx was heavily ubiquitinated by Siah-1 and degraded by the ubiquitin–proteasome system in HepG2 cells, whereas this effect was marginal or undetectable in Hep3B cells. Knock-down of p53 in HepG2 cells downregulated Siah-1 levels and subsequently upregulated HBx levels, whereas ectopic p53 expression in Hep3B cells upregulated Siah-1 levels and subsequently downregulated HBx levels. In addition, Siah-1 knock-down impaired the ubiquitination and proteasomal degradation of HBx in HepG2 cells, whereas ectopic Siah-1 expression induced ubiquitin-dependent proteasomal degradation of HBx in Hep3B cells. The effects of HBx on p53 and Siah-1 were exactly reproduced in a 1.2-mer HBV replicon system, mimicking the natural course of HBV infection. In particular, Siah-1 knock-down upregulated the levels of HBx derived from the HBV replicon, resulting in an increase in HBV production. In conclusion, HBx modulates its own protein level via a negative feedback loop involving p53 and Siah-1 to control HBV propagation.

Adenovirus protein VII is a highly cationic core protein that forms a nucleosome-like structure in the adenovirus core by condensing DNA in combination with protein V and mu. It has been proposed that protein VII could condense DNA in a manner analogous to mammalian histones. Due to the lack of an expression and purification protocol, the interactions between protein VII and DNA are poorly understood. In this study we describe methods for the purification of biologically active recombinant protein VII using an E. coli expression system. We expressed a cleavable fusion of protein VII with thioredoxin and established methods for purification of this fusion protein in denatured form. We describe an efficient method for resolving the cleavage products to obtain pure protein VII using hydroxyapatite column chromatography. Mass spectroscopy data confirmed its mass and purity to be 19.4 kDa and >98 %, respectively. Purified recombinant protein VII spontaneously condensed dsDNA to form particles, as shown by dye exclusion assay, electrophoretic mobility shift assay and nuclease protection assay. Additionally, an in vitro bioluminescence assay revealed that protein VII can be used to enhance the transfection of mammalian cells with lipofectamine/DNA complexes. The availability of recombinant protein VII will facilitate future studies of the structure of the adenovirus core. Improved understanding of the structure and function of protein VII will be valuable in elucidating the mechanism of adenoviral DNA condensation, defining the morphology of the adenovirus core and establishing the mechanism by which adenoviral DNA enters the nucleus.

Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus that causes life-threatening disease in immunocompromised and immunonaïve individuals. Type I interferons (IFNs) are crucial molecules in the innate immune response to HCMV and are also known to upregulate several components of the interchromosomal multiprotein aggregates collectively referred to as nuclear domain 10 (ND10). In the context of herpesvirus infection, ND10 components are known to restrict gene expression. This raises the question as to whether key ND10 components (PML, Sp100 and hDaxx) act as anti-viral IFN-stimulated genes (ISGs) during HCMV infection. In this study, analysis of ND10 component transcription during HCMV infection demonstrated that PML and Sp100 were significantly upregulated whilst hDaxx expression remained unchanged. In cells engineered to block the production of, or response to, type I IFNs, upregulation of PML and Sp100 was not detected during HCMV infection. Furthermore, pre-treatment with an IFN-β neutralizing antibody inhibited upregulation of PML and Sp100 during both infection and treatment with HCMV-infected cell supernatant. The significance of ND10 components functioning as anti-viral ISGs during HCMV infection was determined through knockdown of PML, Sp100 and hDaxx. ND10 knockdown cells were significantly more permissive to HCMV infection, as previously described but, in contrast to control cells, could support HCMV plaque formation following IFN-β pre-treatment. This ability of HCMV to overcome the potently anti-viral effects of IFN-β in ND10 expression deficient cells provides evidence that ND10 component upregulation is a key mediator of the anti-viral activity of IFN-β.

The persistence of African swine fever virus (ASFV) in endemic areas, with small-scale but regular outbreaks in domestic pigs, is not well understood. ASFV has not been detected using conventional diagnosis in these pigs or adjacent populations of resistant African wild pigs, that could act as potential carriers during the outbreaks. However, such data are crucial for the design of evidence-based control strategies. We conducted cross-sectional (1107 pigs) and longitudinal (100 pigs) monitoring of ASFV prevalence in local pigs in Kenya and Uganda. The horizontal survey revealed no evidence of ASFV in the serum or blood using either conventional or real-time PCR. One pig consistently tested positive using ELISA, but negative using PCR assays on blood. Interestingly, the isotype of the antibodies from this animal were strongly IgA biased relative to control domestic pigs and warthogs, suggesting a role for mucosal immunity. The tissues from this pig were positive by PCR following post-mortem. Internal organ tissues of 44 healthy pigs (28 sentinel pigs and 16 pigs from slaughter slabs) were tested with four different PCR assays; 15.9 % were positive for ASFV suggesting that healthy pigs carrying ASFV exist in the swine population in the study area. P72 and p54 genotyping of ASFV revealed very limited diversity: all were classified in genotype IX at both loci, as were virtually all viruses causing recent ASF outbreaks in the region. Our study suggests that carrier pigs may play a role in ASF disease outbreaks, although the triggers for outbreaks remain unclear and require further investigation. This study significantly increases scientific knowledge of the epidemiology of ASF in the field in Africa, which will contribute to the design of effective surveillance and control strategies.

Herpesvirus entry mediator A (HVEM), nectin-1 and nectin-2 are cellular receptors of glycoprotein D (gD) of herpes simplex virus type-2 (HSV-2). It has been shown that soluble forms of HSV gD receptors have the antiviral potential in cultured cells and transgenic mice. Here, to compare antiviral potential of soluble forms of HVEM, nectin-1 and nectin-2 against HSV-2 infections in vivo, transgenic mice expressing fusion proteins consisting of the entire ectodomain of HVEM, nectin-1 or nectin-2 and the Fc portion of human IgG (HVEMIg, nectin-1Ig and nectin-2Ig, respectively) were intraperitoneally infected with HSV-2. In the infection with 3 MLD50 (50 % mouse lethal dose), effective resistance was not observed in transgenic mice expressing nectin-2Ig. In a transgenic mouse line with high expression of nectin-1Ig, significant protection from the infection with 30 and 300 MLD50 was observed (survival rate of 100 and 71 %, respectively). On the other hand, transgenic mice expressing HVEMIg showed a complete resistance to the lethal infection even with 300 MLD50 (survival rate of 100 %). These results demonstrated that HVEMIg could exert effective antiviral activities against HSV-2 infections in vivo as compared with other soluble forms of HSV gD receptors.

Integration of the complete human herpesvirus 6 (HHV-6) genome into the telomere of a chromosome has been reported in some individuals (inherited chromosomally integrated HHV-6; iciHHV-6). Since the proportion of iciHHV-6-positive individuals with integration in chromosome 22 is high in Japan, we hypothesized a founder effect. In this study, we sought to elucidate the reason for the high proportion of viral integrations into chromosome 22. We analyzed six cases of iciHHV-6A and two cases of iciHHV-6B, including one iciHHV-6A case with a matched sample from a father and one iciHHV-6B case with a matched sample from a mother. In iciHHV-6A, the same copy numbers of viral telomeric repeat sequences (TRS) and the same five microsatellite markers were detected in both the index case and paternal sample. Moreover, the same five microsatellite markers were demonstrated in four cases and the same copy numbers of viral TRS were demonstrated in two pairs of two cases. The present microsatellite analysis suggested that the viral genomes detected in some iciHHV-6A patients were derived from a common ancestral integration.

In vitro investigations have identified a variety of mechanisms by which herpesviruses evade interferon-stimulated antiviral effector mechanisms. However, these immune evasion mechanisms have not been evaluated during a bovine herpesvirus-1 (BHV-1) infection. This study investigated the transcription and secretion of type I and II interferons (IFNs) and the transcription of IFN-stimulated genes (ISGs) during a primary BHV-1 infection of the upper respiratory tract (URT) in naïve calves. IFN-α, -β and -γ transcription in nasal turbinates and protein levels in nasal secretions increased following infection. Increased IFN type I and II secretion was detected 3 days post-infection (p.i.) and IFN production increased in parallel with virus shedding. Expression of ISGs, including Mx1, OAS and BST-2, also increased significantly (P<0.05) in nasal turbinates on day 3 p.i. and elevated ISG expression persisted throughout the period of viral shedding. In contrast, RNAase L gene expression was not induced during the BHV-1 infection in the nasal turbinates, but was induced on day 10 p.i. in the trachea. In vitro studies confirmed that recombinant bovine (rBo)IFN-α, -β and -γ induced expression of Mx1, OAS and BST-2, but decreased RNAse L transcript in bovine epithelial cells. Relative to vesicular stomatitisvirus (VSV), BHV-1 was resistant to the antiviral activity of rBoIFN-α and –γ, but treatment of epithelial cells with 10 ng rBoIFN-β ml−1 effected an 80 % inhibition of BHV-1 replication and complete inhibition of VSV replication. These observations confirm that the transcription and translation of type I and II IFNs increase during BHV-1 infection, while the transcription of some ISGs is not inhibited.

In common with other herpes viruses, bovine herpes virus 1 (BHV-1) induces strong virus-specific CD8 T-cell responses. However, there is a paucity of information on the antigenic specificity of the responding T-cells. The development of a system to generate virus-specific CD8 T-cell lines from BHV-1-immune cattle, employing Theileria-transformed cell lines for antigen presentation, has enabled us to address this issue. Use of this system allowed the study to screen for CD8 T-cell antigens that are efficiently presented on the surface of virus-infected cells. Screening of a panel of 16 candidate viral gene products with CD8 T-cell lines from 3 BHV-1-immune cattle of defined MHC genotypes identified 4 antigens, including 3 immediate early (IE) gene products (ICP4, ICP22 and Circ) and a tegument protein (UL49). Identification of the MHC restriction specificities revealed that the antigens were presented by two or three class I MHC alleles in each animal. Six CD8 T-cell epitopes were identified in the three IE proteins by screening of synthetic peptides. Use of an algorithm (NetMHCpan) that predicts the peptide-binding characteristics of restricting MHC alleles confirmed and, in some cases refined, the identity of the epitopes. Analyses of the epitope specificity of the CD8 T-cell lines showed that a large component of the response is directed against these IE epitopes. The results indicate that these IE gene products are dominant targets of the CD8 T-cell response in BHV-I-immune cattle and hence are prime-candidate antigens for the generation of a subunit vaccine.

The level of antioxidants, such as thiol-containing tripeptide glutathione (GSH), in cytomegalovirus (CMV)-infected cells is notably increased. We previously showed that GSH levels in plasma, as measured by untargeted 1H nuclear magnetic resonance, are higher in allogeneic stem cell transplant (allo-SCT) recipients who subsequently develop CMV viraemia. We hypothesized that the net level of oxidative-stress markers present in plasma may be reduced in patients who develop CMV DNAaemia compared to those who do not. We serially monitored the levels of malondialdehyde (MDA) and carbonylated proteins (CPs) early after allo-SCT and assessed whether they could predict the occurrence of CMV DNAaemia. MDA levels were measured in 43 patients (28 had CMV DNAaemia) and CPs were quantified in 53 patients (38 patients developed CMV DNAaemia). The area under the curve (AUC) value for MDA, but not for CPs, was significantly lower in patients who subsequently developed CMV DNAaemia compared to those who remained DNAaemia-free (P=0.043). A trend toward lower MDA AUC values was observed in episodes of CMV DNAaemia with faster CMV replicative kinetics and in those who reached higher peak CMV DNA levels. Moreover, receiver operating characteristic curve analyses indicated that the MDA biomarker had the predictive ability to discriminate between patients with or without subsequent CMV DNAaemia (AUC=0.69, 95 % confidence interval 0.51–0.85, P=0.05). In summary, serial quantitation of MDA may be useful for individualizing antiviral prophylaxis therapies (targeted prophylaxis) in the upcoming era of new antiviral drugs with improved safety profiles.

The connection between the repression of human immunodeficiency virus type 1(HIV-1) transcription and the resting CD4+ T cell state suggests that the host transcription factors involved in the active maintenance of lymphocyte quiescence are likely to repress the viral transactivator, Tat, thereby restricting HIV-1 transcription. In this study, we analysed the interplay between Tat and the forkhead box transcription factors, FoxO1 and FoxO4. We show that FoxO1 and FoxO4 antagonize Tat-mediated transactivation of HIV-1 promoter through the repression of Tat protein expression. No effect was observed on the expression of two HIV-1 accessory proteins, Vif and Vpr. Unexpectedly, we found that FoxO1 and FoxO4 expression causes a strong dose-dependent post-transcriptional suppression of Tat mRNA, indicating that FoxO should effectively inhibit HIV-1 replication by destabilizing Tat mRNA and suppressing Tat-mediated HIV-1 transcription. In accordance with this, we observed that the Tat mRNA half-life is reduced by FoxO4 expression. The physiological relevance of our findings was validated using the J-Lat 10.6 model of latently infected cells. We demonstrated that the overexpression of a constitutively active FoxO4-TM mutant antagonized HIV-1 transcription reactivation in response to T cell activators, such as TNF-α or PMA. Altogether, our findings demonstrate that FoxO factors can control HIV-1 transcription and provide new insights into their potential role during the establishment of HIV-1 latency.

Delineating the factors leading to the development of broadly neutralizing antibodies (bnAbs) during natural HIV-1 infection and dissecting their epitope specificities generates useful information for vaccine design. This is the first longitudinal study to assess the plasma-neutralizing antibody response and neutralizing determinants in HIV-1-infected children from India. We enrolled 26 and followed up 20 antiretroviral therapy (ART)-naïve, asymptomatic, chronic HIV-1-infected children. Five (19.2 %) baseline and 10 (50 %) follow-up plasma samples neutralized ≥50 % of subtypes A, B and C tier 2 viruses at an ID50 titre ≥150. A modest improvement in neutralization breadth and potency was observed with time. At baseline, subtype C-specific neutralization predominated (P=0.026); interestingly, follow-up samples exhibited cross-neutralizing activity. Epitope mapping revealed V3C reactive antibodies with significantly increased Max50 binding titres in follow-up samples from five infected children; patient #4's plasma antibodies exhibited V3-directed neutralization. A salient observation was the presence of CD4 binding site (CD4bs)-specific NAbs in patient #18 that improved with time (1.76-fold). The RSC3 wild-type (RSC3WT) protein-depleted plasma eluate of patient #18 demonstrated a more than 50% ID50 decrease in neutralization capacity against five HIV-1 pseudoviruses. Further, the presence of CD4bs-neutralizing determinants in patient #18's plasma was confirmed by the neutralizing activity demonstrated by the CD4bs-directed IgG fraction purified from this plasma, and competition with sCD4 against JRFLgp120, identifying this paediatric donor as a potential candidate for the isolation of CD4bs-directed bnAbs. Overall, we observed a relative increase in plasma-neutralizing activity with time in HIV-1-infected children, which suggests that the bnAbs evolve.

MicroRNAs (miRNAs) are important regulators of biological processes, including host–virus interaction. This study investigated the involvement of Drosophila melanogaster miR-8-5p in host–virus interaction. Drosophila flies and cells challenged with Drosophila C virus (DCV) were found to have lower miR-8-5p abundance compared to uninfected samples. Lowering miR-8-5p abundance by experimental inhibition of the miRNA led to an increase in viral accumulation, suggesting that the observed decrease in the miR-8-5p abundance during DCV infection enhances viral replication. miR-8-5p putative targets were identified and included dJun, a transcription factor gene whose mammalian homologue cJun is induced by various viruses through kinase activation. Increasing miR-8-5p abundance using miR-8-5p mimics resulted in a decrease in dJun and GFP reporter levels. Furthermore, when the putative target in dJun was mutated, addition of miR-8-5p mimics did not result in the same antagonistic effect on dJun. These results show negative regulation of dJun by miR-8-5p and suggest that an miRNA-mediated pathway is involved in dJun regulation during viral infection. To analyse the role of dJun during DCV infection, dJun was knocked down in cells prior to DCV infection. Knockdown of dJun decreased DCV replication, providing evidence that dJun up-regulation that is concomitant with miR-8-5p down-regulation during DCV infection supports viral replication. These results highlight the role of miRNA in regulating the transcription factor gene dJun and uncover a previously unrecognized mechanism by which dJun is regulated during host–virus interaction.

Infection of plants by viruses is a complex process involving several steps: inoculation into plant cells, replication in inoculated cells and plant colonization. The success of the different steps depends, in part, on the viral effective population size (Ne ), defined as the number of individuals passing their genes to the next generation. During infection, the virus population will undergo bottlenecks, leading to drastic reductions in Ne and, potentially, to the loss of the fittest variants. Therefore, it is crucial to better understand how plants affect Ne . We aimed to (i) identify the plant genetic factors controlling Ne during inoculation, (ii) understand the mechanisms used by the plant to control Ne and (iii) compare these genetic factors with the genes controlling plant resistance to viruses. Ne was measured in a doubled-haploid population of Capsicum annuum. Plants were inoculated with either a Potato virus Y (PVY) construct expressing the green fluorescent protein or a necrotic variant of Cucumber mosaic virus (CMV). Ne was assessed by counting the number of primary infection foci on cotyledons for PVY or the number of necrotic local lesions on leaves for CMV. The number of foci and lesions was correlated (r=0.57) and showed a high heritability (h2 =0.93 for PVY and h2 =0.98 for CMV). The Ne of the two viruses was controlled by both common quantitative trait loci (QTLs) and virus-specific QTLs, indicating the contribution of general and specific mechanisms. The PVY-specific QTL colocalizes with a QTL that reduces PVY accumulation and the capacity to break down a major-effect resistance gene.

Prion diseases are a unique group of transmissible, typically sub-acute, neurodegenerative disorders. During central nervous system (CNS) prion disease, the microglia become activated and are thought to provide a protective response by scavenging and clearing prions. The mammalian intestine is host to a large burden of commensal micro-organisms, especially bacteria, termed the microbiota. The commensal microbiota has beneficial effects on host health, including through the metabolism of essential nutrients, regulation of host development and protection against pathogens. The commensal gut microbiota also constitutively regulates the functional maturation of microglia in the CNS, and microglial function is impaired when it is absent in germ-free mice. In the current study, we determined whether the absence of the commensal gut microbiota might also affect prion disease pathogenesis. Our data clearly show that the absence of the commensal microbiota in germ-free mice did not affect prion disease duration or susceptibility after exposure to prions by intraperitoneal or intracerebral injection. Furthermore, the magnitude and distribution of the characteristic neuropathological hallmarks of terminal prion disease in the CNS, including the development of spongiform pathology, accumulation of prion disease-specific protein (PrP), astrogliosis and microglial activation, were similar in conventionally housed and germ-free mice. Thus, although the commensal gut microbiota constitutively promotes the maintenance of the microglia in the CNS under steady-state conditions in naïve mice, our data suggest that dramatic changes to the abundance or complexity of the commensal gut microbiota are unlikely to influence CNS prion disease pathogenesis.

Chronic wasting disease (CWD) is an emergent prion disease affecting cervid species in North America, Canada, South Korea, and recently, Norway. Detection of CWD has been advanced by techniques that rely on amplification of low levels of prion amyloid to a detectable level. However, the increased sensitivity of amplification assays is often compromised by inhibitors and/or activators in complex biologic samples including body fluids, excreta, or the environment. Here, we adapt real-time quaking-induced conversion conditions to specifically detect CWD prions in fecal samples from both experimentally infected deer and naturally infected elk and estimate environmental contamination. The results have application to detection, surveillance and management of CWD, and potentially to other protein-misfolding diseases.