- Volume 96, Issue 1, 2015

A common critical cellular event that many human enveloped viruses share is the requirement for proteolytic cleavage of the viral glycoprotein by furin in the host secretory pathway. For example, the furin-dependent proteolytic activation of highly pathogenic (HP) influenza A (infA) H5 and H7 haemagglutinin precursor (HA0) subtypes is critical for yielding fusion-competent infectious virions. In this study, we hypothesized that viral hijacking of the furin pathway by HP infA viruses to permit cleavage of HA0 could represent a novel molecular mechanism controlling the dynamic production of fusion-competent infectious virus particles during the viral life cycle. We explored the biological role of a newly identified furin-directed human microRNA, miR-24, in this process as a potential post-transcriptional regulator of the furin-mediated activation of HA0 and production of fusion-competent virions in the host secretory pathway. We report that miR-24 and furin are differentially expressed in human A549 cells infected with HP avian-origin infA H5N1. Using miR-24 mimics, we demonstrated a robust decrease in both furin mRNA levels and intracellular furin activity in A549 cells. Importantly, pretreatment of A549 cells with miR-24 mimicked these results: a robust decrease of H5N1 infectious virions and a complete block of H5N1 virus spread that was not observed in A549 cells infected with low-pathogenicity swine-origin infA H1N1 virus. Our results suggest that viral-specific downregulation of furin-directed microRNAs such as miR-24 during the life cycle of HP infA viruses may represent a novel regulatory mechanism that governs furin-mediated proteolytic activation of HA0 glycoproteins and production of infectious virions.

Newcastle disease virus (NDV) has been developed as a vector for vaccine and gene therapy purposes. However, the optimal insertion site for foreign gene expression remained to be determined. In the present study, we inserted the green fluorescence protein (GFP) gene into five different intergenic regions of the enterotropic NDV VG/GA vaccine strain using reverse genetics technology. The rescued recombinant viruses retained lentogenic pathotype and displayed delayed growth dynamics, particularly when the GFP gene was inserted between the NP and P genes of the virus. The GFP mRNA level was most abundant when the gene was inserted closer to the 3′ end and gradually decreased as the gene was inserted closer to the 5′ end. Measurement of the GFP fluorescence intensity in recombinant virus-infected cells demonstrated that the non-coding region between the P and M genes is the optimal insertion site for foreign gene expression in the VG/GA vaccine vector.

The outbreak of human infections caused by the novel avian-origin H7N9 influenza viruses in China since March 2013 underscores the urgent need to find an effective treatment strategy against H7N9 infection in humans. In this study, we assessed the effectiveness of combinations of oseltamivir and two immunomodulators (simvastatin and fenofibrate) against H7N9 infection in a mouse model. Mice treated with oseltamivir plus fenofibrate exhibited the longest mean survival time, the largest reduction of viral titre in lung tissue, the highest levels of CD4+ and CD8+ T-lymphocytes, and the greatest decrease in pulmonary inflammation. Thus, the combination of oseltamivir plus fenofibrate improved the outcomes of mice infected with H7N9 virus by simultaneously reducing viral replication and normalizing the aberrant immune response. This drug combination should be considered in randomized controlled trials of treatments for H7N9 patients.

The antibody response plays a crucial role against hepatitis C virus (HCV) infection, and our understanding of this intricate progress in vivo is far from complete. We previously reported a novel and robust technique based on a large combinatorial viral antigen library displayed on the surface of the yeast Saccharomyces cerevisiae, allowing comprehensive profiling of polyclonal antibody responses in vivo in both qualitative and quantitative terms. Here, we report the generation and application of a combinatorial library of HCV strain JFH1 envelope glycoprotein to profile the antibody response in four HCV chronically infected individuals. By systematic analysis of the location and frequency of antigenic fragments along the JFH1 envelope glycoprotein, we showed that the major binding antibody response was targeted to E2 (80.9–99.8 %), whilst that against E1 was relatively small (0.3–19.0 %). A total of five major antigenic domains (D1–D5) were identified: one was within E1 and an additional four within E2, despite substantial variability among the different individuals. However, serum absorption with the yeast clones containing the antigenic domain D1 resulted in more reduction in neutralizing antibody activity against pseudotyped HCV than those in E2, suggesting that E1 contains additional neutralizing epitopes. Our results have provided additional insights into the HCV-specific antibody response in humans and should assist in a better understanding of protective antibody immunity and in guiding the development of effective vaccines and therapeutics against HCV infection.

In contrast to the current wealth of structural information concerning dicistrovirus particle structure, very little is known about their morphogenetic pathways. Here, we describe the expression of the two ORFs encoded by the Triatoma virus (TrV) genome. TrV, a member of the Cripavirus genus of the Dicistroviridae family, infects blood-sucking insects belonging to the Triatominae subfamily that act as vectors for the transmission of Trypanosoma cruzi, the aetiological agent of the Chagas disease. We have established a baculovirus-based model for the expression of the NS (non-structural) and P1 (structural) polyproteins. A preliminary characterization of the proteolytic processing of both polyprotein precursors has been performed using this system. We show that the proteolytic processing of the P1 polyprotein is strictly dependent upon the coexpression of the NS polyprotein, and that NS/P1 coexpression leads to the assembly of virus-like particles (VLPs) exhibiting a morphology and a protein composition akin to natural TrV empty capsids. Remarkably, the unprocessed P1 polypeptide assembles into quasi-spherical structures conspicuously larger than VLPs produced in NS/P1-coexpressing cells, likely representing a previously undescribed morphogenetic intermediate. This intermediate has not been found in members of the related Picornaviridae family currently used as a model for dicistrovirus studies, thus suggesting the existence of major differences in the assembly pathways of these two virus groups.

Porcine sapovirus (PSaV) of the family Caliciviridae, is the only member of the genus Sapovirus with cell culture and reverse genetics systems. When combined with the piglet model, these approaches provide a system to understand the molecular basis of sapovirus pathogenesis. The replication of PSaV in cell culture is, however, restricted, displaying an absolute requirement for bile acids and producing lower levels of infectious virus than other caliciviruses. The effect of bile acids has previously been linked to a reduction in the signal transducer and activator of transcription (STAT1)-mediated signalling pathway. In the current study, we observed that even in the presence of bile acids, PSaV replication in cell culture was restricted by soluble factors produced from infected cells. This effect was at least partially due to secreted IFN because treatment of cells with recombinant porcine IFN-β resulted in significantly reduced viral replication. Moreover, IFN-mediated signalling pathways (IFN, STAT1 and the 2′,5′-oligoadenylate synthetase) were activated during PSaV infection. Characterization of PSaV growth in cell lines deficient in their ability to induce or respond to IFN showed a 100–150-fold increase in infectious virus production, indicating that the primary role of bile acids was not the inactivation of the innate immune response. Furthermore, the use of IFN-deficient cell lines enabled more efficient recovery of PSaV from cDNA constructs. Overall, the highly efficient cell culture and reverse genetics system established here for PSaV highlighted the key role of the innate immune response in the restriction of PSaV infection and should greatly facilitate further molecular studies on sapovirus host–cell interactions.

MicroRNAs (miRNAs) are a class of short endogenous RNA molecules with the ability to control development, autophagy, apoptosis and the stress response in eukaryotes by pairing with partially complementary sites in the 3′ UTRs of targeted genes. Recent studies have demonstrated that miRNAs serve as critical effectors in intricate networks of host–pathogen interactions. Notably, we found that Bos taurus bta-miR-29b (referred to as miR-29b herein) was significantly upregulated >2.3-fold in bovine viral diarrhoea virus (BVDV) strain NADL-infected Madin–Darby bovine kidney (MDBK) cells 6 h post-infection compared with normal MDBK cells. However, the roles of miR-29b in BVDV infection and pathogenesis remain unclear. Here, we report the inhibitory effects of miR-29b on BVDV NADL replication and viral infection-related autophagy. miR-29b overexpression mediated by miRNA precursor-expressing lentivirus resulted in the attenuation of BVDV NADL infection-related autophagy by directly downregulating the intracellular expression levels of two key autophagy-associated proteins, ATG14 and ATG9A. Moreover, ATG14 and ATG9A overexpression rescue not only reversed miR-29b-inhibited autophagy, but also increased BVDV NADL replication. In previous studies, we found that the early stages of autophagy contributed to BVDV NADL replication in MDBK cells and that the inhibition of autophagy repressed BVDV NADL replication, which was also proved in the present study. Collectively, our results establish a novel link between miR-29b and viral replication, and also provide a new pathway for the intimate interaction between host cells and pathogens.

Cloning and sequencing of Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) genome segment S4 showed that it consists of 3410 nt with a single ORF of 1110 aa which could encode a protein of ~127 kDa (p127). Bioinformatics analysis showed the presence of a 5′ RNA triphosphatase (RTPase) domain (LRDR), a S-adenosyl-l-methionine (SAM)-binding (GxGxG) motif and the KDKE tetrad of 2′-O-methyltransferase (MTase), which suggested that S4 may encode RTPase and MTase. The ORF of S4 was expressed in Escherichia coli as a His-tagged fusion protein and purified by nickel-nitrilotriacetic acid affinity chromatography. Biochemical analysis of recombinant p127 showed its RTPase as well as SAM-dependent guanine N 7-and ribose 2′-O-MTase activities. A MTase assay using in vitro transcribed AmCPV S2 RNA having a 5′ G*pppG end showed that guanine N 7 methylation occurred prior to the ribose 2′-O methylation to yield a m7GpppG/m7GpppGm RNA cap. Mutagenesis of the SAM-binding (GxGxG) motif (G831A) completely abolished N 7- and 2′-O-MTase activities, indicating the importance of these residues for capping. From the kinetic analysis, the K m values of N 7-MTase for SAM and RNA were calculated as 4.41 and 0.39 µM, respectively. These results suggested that AmCPV S4-encoded p127 catalyses RTPase and two cap methylation reactions for capping the 5′ end of viral RNA.

Group A rotaviruses (RVAs) are enteric pathogens with well-documented zoonotic transmissions to humans. The segmented genome of the virus enables reassortment events which might alter host susceptibility and/or disease course. Genetic analysis of rotavirus in dogs has so far only revealed RVAs with the VP7 and VP4 genome constellation G3P[3]. RVA G3P[3] have also been found in cats, humans, monkeys and bats. In the present study, we described an unusual RVA of genotype G8P[1] which was isolated from an asymptomatically infected young dog. The dog did not show signs of diarrhoea. Analysis of full-length segments of VP2, VP6 and VP7 as well as NSP1–NSP5 revealed a typical bovine-like genotype constellation G8–P[1]–I2–Rx–C2–Mx–A3–N2–T6–E2–H3. Phylogenetic analysis supported the hypothesis of an interspecies transmission from a bovine/artiodactyl species or from humans to the young dog. The isolate was likely to represent a multiple reassortant virus.

Torque teno virus (TTV) is increasingly considered a universal marker of global immune function. The virus is supposed to replicate in lymphocytes, but poor information is available about fluctuations of viraemia after administration of anti-lymphocyte agents. We studied TTV kinetics in a cohort of 70 kidney±pancreas recipients receiving one of two different anti-T-cell induction immunosuppressants. During the first 30 days after anti-T-cell antibody administration, we report kinetics of TTV viraemia compatible with replication in T lymphocytes, and highly dependent on the potency of the anti-T-cell drug administered.

Equine herpesvirus type 1 (EHV-1) replicates in the epithelial cells of the upper respiratory tract and disseminates through the body via a cell-associated viraemia in monocytic cells, despite the presence of neutralizing antibodies. However, the mechanism by which EHV-1 hijacks immune cells and uses them as ‘Trojan horses’ in order to disseminate inside its host is still unclear. Here, we hypothesize that EHV-1 delays its replication in monocytic cells in order to avoid recognition by the immune system. We compared replication kinetics in vitro of EHV-1 in RK-13, a cell line fully susceptible to EHV-1 infection, and primary horse cells from the myeloid lineage (CD172a+). We found that EHV-1 replication was restricted to 4 % of CD172a+ cells compared with 100 % in RK-13 cells. In susceptible CD172a+ cells, the expression of immediate-early (IEP) and early (EICP22) proteins was delayed in the cell nuclei by 2–3 h post-infection (p.i.) compared with RK-13, and the formation of replicative compartments by 15 h p.i. Virus production in CD172a+ cells was significantly lower (from 101.7 to 103.1 TCID50 per 105 inoculated cells) than in RK-13 (from 105 to 105.7 TCID50 per 105 inoculated cells). Less than 0.02 % of inoculated CD172a+ cells produced and transmitted infectious virus to neighbouring cells. Pre-treatment of CD172a+ cells with inhibitors of histone deacetylase activity increased and accelerated viral protein expression at very early times of infection and induced productive infection in CD172a+ cells. Our results demonstrated that the restriction and delay of EHV-1 replication in CD172a+ cells are part of an immune evasive strategy and involve silencing of EHV-1 gene expression associated with histone deacetylases.

Graft rejection in transplant patients is managed clinically by suppressing T-cell function with immunosuppressive drugs such as prednisolone and methylprednisolone. In such immunocompromised hosts, human cytomegalovirus (HCMV) is an important opportunistic pathogen and can cause severe morbidity and mortality. Currently, the effect of glucocorticosteroids (GCSs) on the HCMV life cycle remains unclear. Previous reports showed enhanced lytic replication of HCMV in vitro in the presence of GCSs. In the present study, we explored the implications of steroid exposure on latency and reactivation. We observed a direct effect of several GCSs used in the clinic on the activation of a quiescent viral major immediate-early promoter in stably transfected THP-1 monocytic cells. This activation was prevented by the glucocorticoid receptor (GR) antagonist Ru486 and by shRNA-mediated knockdown of the GR. Consistent with this observation, prednisolone treatment of latently infected primary monocytes resulted in HCMV reactivation. Analysis of the phenotype of these cells showed that treatment with GCSs was correlated with differentiation to an anti-inflammatory macrophage-like cell type. On the basis that these observations may be pertinent to HCMV reactivation in post-transplant settings, we retrospectively evaluated the incidence, viral kinetics and viral load of HCMV in liver transplant patients in the presence or absence of GCS treatment. We observed that combination therapy of baseline prednisolone and augmented methylprednisolone, upon organ rejection, significantly increased the incidence of HCMV infection in the intermediate risk group where donor and recipient are both HCMV seropositive (D+R+) to levels comparable with the high risk D+R− group.

Infection of astrocytes by the neuropathogenic mutant of Moloney murine leukemia virus, ts1, exhibits increased levels of reactive oxygen species (ROS) and signs of oxidative stress compared with uninfected astrocytes. Previously, we have demonstrated that ts1 infection caused two separate events of ROS upregulation. The first upregulation occurs during early viral establishment in host cells and the second during the virus-mediated apoptotic process. In this study, we show that virus-mediated ROS upregulation activates the protein kinase, ataxia telangiectasia mutated, which in turn phosphorylates serine 15 on p53. This activation of p53 however, is unlikely associated with ts1-induced cell death. Rather p53 appears to be involved in suppressing intracellular ROS levels in astrocytes under oxidative stress. The activated p53 appears to delay retroviral gene expression by suppressing NADPH oxidase, a superoxide-producing enzyme. These results suggest that p53 plays a role as a retrovirus-mediated oxidative stress modulator.

Avian leukosis virus subgroup J (ALV-J) was first isolated from meat-producing chickens that had developed myeloid leukosis. However, ALV-J infections associated with hemangiomas have occurred in egg-producing (layer) flocks in China. In this study, we identified an ALV-J layer isolate (HLJ13SH01) as a recombinant of ALV-J and a Rous sarcoma virus Schmidt-Ruppin B strain (RSV-SRB), which contained the RSV-SRB 5′-LTR and the other genes of ALV-J. Replication kinetic testing indicated that the HLJ13SH01 strain replicated faster than other ALV-J layer isolates in vitro. Sequence analysis indicated that the main difference between the two isolates was the 5′-LTR sequences, particularly the U3 sequences. A 19 nt insertion was uniquely found in the U3 region of the HLJ13SH01 strain. The results of a Dual-Glo luciferase assay revealed that the 19 nt insertion in the HLJ13SH01 strain increased the enhancer activity of the U3 region. Moreover, an additional CCAAT/enhancer element was found in the 19 nt insertion and the luciferase assay indicated that this element played a key role in increasing the enhancer activity of the 5′-U3 region. To confirm the potentiation effect of the 19 nt insertion and the CCAAT/enhancer element on virus replication, three infectious clones with 5′-U3 region variations were constructed and rescued. Replication kinetic testing of the rescued viruses demonstrated that the CCAAT/enhancer element in the 19 nt insertion enhanced the replication capacity of the ALV-J recombinant in vitro.

Human T-cell leukaemia virus type-1 (HTLV-1) infection causes adult T-cell leukaemia (ATL). The viral protein HTLV-1 bZIP factor (HBZ) is constitutively expressed in ATL cells, suggesting that HBZ plays a major role in the pathogenesis of HTLV-1-associated disease. Here, we identified centromere protein B (CENP-B) as a novel interacting partner of HBZ. HBZ and CENP-B associate with their central regions in cells. Furthermore, overexpression of HBZ abrogated the DNA-binding activity of CENP-B to the α-satellite DNA region containing the CENP-B box motif, which in turn inhibited the CENP-B-mediated trimethylation of histone H3K9 in T-cells.