Enhanced Virus Replication Research Articles

184: Glucocorticosteroids dampen anti-viral defences and enhance the replication of respiratory viruses

Aims Viral upper respiratory tract infections caused by rhinovirus (RV) and influenza A virus (IAV) are associated with 50–80% of asthma exacerbations. Inhaled glucocorticosteroids (GCS) are routinely used to relieve asthma symptoms, and while it has been widely reported that GCS reduce inflammation associated with viral infection, the effects of GCS on viral replication has not been comprehensively examined. Methods Using primary human airway cells and a mouse model of IAV infection we examined the effect of GCS on the production of inflammatory mediators, anti-viral responses and viral replication following RV and/or IAV infection. Results GCS pre-treatment of epithelial cells significantly reduced IL-8, IL-6 and IP-10 production following RV and IAV infection, compared to untreated controls. The expression of anti-viral genes, including ISG56, ISG15, viperin and MxA, were significantly reduced in epithelial cells, fibroblasts and macrophages pre-treated with GCS prior to infection with RV or IAV. The impairment of anti-viral responses observed following GCS pre-treatment was associated with enhanced RV and IAV replication in both epithelial cells and fibroblasts. GCS pre-treatment of mice prior to IAV infection was associated with significant weight loss and severe disease progression compared with untreated mice. Viral loads were significantly elevated in both the lungs and nasal tissues of the GCS treated mice, and there were significantly elevated levels of IL-6, MCP-1 and KC in BAL fluid. Interferon and anti-viral ISG expression were significantly reduced in GCS treated mice. Treatment with recombinant interferon after infection delayed the onset of disease and improved survival. Conclusions Our data indicate that GCS at therapeutic doses enhanced virus replication through the suppression of innate anti-viral responses. Interferon treatment improved disease severity in the mouse model, suggesting this may be a viable therapeutic option to counteract the adverse effects of GCS treatment on viral replication and innate immune suppression.

Characterization of the Polyproline Region of the Hepatitis E Virus in Immunocompromised Patients

Little is known about virus adaptation in immunocompromised patients with chronic genotype 3 hepatitis E virus (HEV3) infections. Virus-host recombinant strains have been isolated recently from chronically infected patients. The nature and incidence of such recombinant events occurring during infections of solid-organ transplant (SOT) recipients are essentially unknown. The polyproline region (PPR) of strains isolated from SOT patients was sequenced during the acute-infection phase (n = 59) and during follow-up of patients whose infections became chronic (n = 27). These 27 HEV strains included 3 (11%) that showed recombinant events 12, 34, 48, or 88 months after infection. In one strain, parts of the PPR and the RNA-dependent RNA polymerase were concomitantly inserted. In the second, a fragment of a human tyrosine aminotransferase (TAT) gene was inserted first, followed by a fragment of PPR. A fragment of the human inter-α-trypsin inhibitor (ITI) gene was inserted in the third. All the inserted sequences were rich in aliphatic and basic amino acids. In vitro growth experiments suggest that the ITI insertion promoted more vigorous virus growth. In silico studies showed that the inserted sequences could provide potential acetylation, ubiquitination, and phosphorylation sites. We found that recombinant events had occurred in the HEV PPR in approximately 11% of the strains isolated from chronically infected transplant patients followed up in Toulouse University Hospital. These inserted fragments came from the HEV genome or a human gene and could enhance virus replication. Importance: Hepatitis E virus (HEV) can cause chronic infections in immunocompromised patients, including solid-organ transplant (SOT) recipients. Two strains that had undergone recombination with human ribosomal genes were described recently. The strains with inserted sequences replicated better in vitro. Little is known about the frequency of such recombinant events or how such an insertion enhances replication. We therefore investigated 59 SOT patients infected with HEV and found 3 strains with 4 recombinant events in 27 of these patients whose infection became chronic. The 4 inserted sequences were of different origins (human gene or HEV genome), but all were enriched in aliphatic and basic amino acids and provided potential regulation sites. Our data indicate that recombinant events occur in approximately 11% of strains isolated from chronically infected patients. The structures of the inserted sequences provide new clues as to how the inserted sequences could foster virus replication.

RETRACTED: Modified Newcastle disease virus vectors expressing the H5 hemagglutinin induce enhanced protection against highly pathogenic H5N1 avian influenza virus in chickens

Naturally-occurring attenuated strains of Newcastle disease virus (NDV) are being developed as vaccine vectors for use in poultry and humans. However, some NDV strains, such as Beaudette C (BC), may retain too much virulence in poultry for safe use, and more highly attenuated strains may be suboptimally immunogenic. We therefore modified the BC strain by changing the multibasic cleavage site sequence of the F protein to the dibasic sequence of avirulent strain LaSota. Additionally, the BC, F, and HN proteins were modified in several ways to enhance virus replication. These modified BC-derived vectors and the LaSota strain were engineered to express the hemagglutin (HA) protein of H5N1 highly pathogenic influenza virus (HPAIV). In general, the modified BC-based vectors expressing HA replicated better than LaSota/HA, and expressed higher levels of HA protein. Pathogenicity tests indicated that all the modified viruses were highly attenuated in chickens. Based on in vitro characterization, two of the modified BC vectors were chosen for evaluation in chickens as vaccine vectors against H5N1 HPAIV A/Vietnam/1203/04. Immunization of chickens with rNDV vector vaccines followed by challenge with HPAIV demonstrated high levels of protection against clinical disease and mortality. However, only those chickens immunized with modified BC/HA in which residues 271-330 from the F protein had been replaced with the corresponding sequence from the NDV AKO strain conferred complete protection against challenge virus shedding. Our findings suggest that this modified rNDV can be used safely as a vaccine vector with enhanced replication, expression, and protective efficacy in avian species, and potentially in humans.

Antiviral Activity of Human OASL Protein Is Mediated by Enhancing Signaling of the RIG-I RNA Sensor

Virus infection is sensed in the cytoplasm by retinoic acid-inducible gene I (RIG-I, also known as DDX58), which requires RNA and polyubiquitin binding to induce type I interferon (IFN) and activate cellular innate immunity. We show that the human IFN-inducible oligoadenylate synthetases-like (OASL) protein has antiviral activity and mediates RIG-I activation by mimicking polyubiquitin. Loss of OASL expression reduced RIG-I signaling and enhanced virus replication in human cells. Conversely, OASL expression suppressed replication of a number of viruses in a RIG-I-dependent manner and enhanced RIG-I-mediated IFN induction. OASL interacted and colocalized with RIG-I, and through its C-terminal ubiquitin-likedomain specifically enhanced RIG-I signaling. Bone-marrow-derived macrophages from mice deficient for Oasl2 showed that among the two mouse orthologs of human OASL, Oasl2 is functionally similar to human OASL. Our findings show a mechanism by which human OASL contributes to host antiviral responses by enhancing RIG-I activation.

Importin-α7 is required for enhanced influenza A virus replication in the alveolar epithelium and severe lung damage in mice.

Influenza A viruses recruit components of the nuclear import pathway to enter the host cell nucleus and promote viral replication. Here, we analyzed the role of the nuclear import factor importin-α7 in H1N1 influenza virus pulmonary tropism by using various ex vivo imaging techniques (magnetic resonance imaging, confocal laser scanning microscopy, and correlative light-electron microscopy). We infected importin-α7 gene-deficient (α7(-/-)) mice with a recombinant H1N1 influenza virus and compared the in vivo viral kinetics with those in wild-type (WT) mice. In WT mice, influenza virus replication in the bronchial and alveolar epithelium already occurred a few days after infection. Accordingly, extensive mononuclear infiltration and alveolar destruction were present in the lungs of infected WT mice, followed by 100% lethality. Conversely, in α7(-/-) mice, virus replication was restricted mostly to the bronchial epithelium with marginal alveolar infection, resulting in significantly reduced lung damage and enhanced animal survival. To investigate the host immune response during alveolar virus replication, we studied the role of primary macrophages in virus propagation and clearance. The ability of macrophages to support or clear the virus infection, as well as the host cellular immune responses, did not significantly differ between WT and α7(-/-) mice. However, cytokine and chemokine responses were generally elevated in WT mice, likely reflective of increased viral replication in the lung. In summary, these data show that a cellular factor, importin-α7, is required for enhanced virus replication in the alveolar epithelium, resulting in elevated cytokine and chemokine levels, extensive mononuclear infiltration, and thus, severe pneumonia and enhanced virulence in mice. Importance: Influenza A viruses are respiratory pathogens that may cause pneumonia in humans. Viral infection and replication in the alveoli of the respiratory tract are believed to be crucial for the development of the acute respiratory distress syndrome associated with fatal outcomes in influenza virus-infected patients. Here, we report the requirement of a cellular factor, importin-α7, for efficient virus replication in the alveolar epithelium of mice. Using complementary ex vivo imaging approaches, we show that influenza virus replication is restricted to the bronchial epithelium, followed by enhanced survival in importin-α7-deficient mice. In contrast, the presence of this gene results in enhanced virus replication in the alveoli, elevated cytokine and chemokine responses, mononuclear infiltration, alveolar destruction, and 100% lethality in wild-type mice. Taken together, our results show that importin-α7 is particularly required for virus replication in the alveolar epithelium in association with severe pneumonia and death in mice.

Antiviral activity of human OASL is mediated by mimicking ubiquitin chains to activate RIG-I pathway (INM6P.421)

Abstract Virus infection is sensed in the cytoplasm by RIG-I, which requires RNA and poly-ubiquitin binding to induce type I interferon (IFN), and activate cellular innate immunity. We show that unlike mouse, the human IFN-inducible oligoadenylate synthetases-like (OASL) protein has antiviral activity and mediates RIG-I activation by mimicking polyubiquitin. Loss of OASL expression reduces RIG-I signaling and enhances virus replication in human cells. Conversely, OASL expression suppresses replication of a number of viruses in a RIG-I-dependent manner and enhances RIG-I-mediated IFN induction. OASL interacts and colocalizes with RIG-I, and through its C-terminal ubiquitin-like domain specifically enhances RIG-I signaling. Importantly, inactive RIG-I mutants defective in ubiquitination and polyubiquitin binding still bind to OASL, and their signaling activity is rescued by OASL. Our findings show a novel mechanism by which human OASL contributes to host antiviral responses by enhancing RIG-I activation.

New Insights Into the Relationships Among Alcohol Consumption, Hepatocellular Carcinoma and Hepatitis C Virus Infection

Context: Viral hepatitis and the consumption of alcohol are recognized as important reasons for the development of liver disease throughout the world. It would also seem that chronic alcoholism causes more severe and rapid progression of liver disease in patients with chronic hepatitis C, leading to more frequent liver cirrhosis and hepatocellular carcinoma. Evidence Acquisition: The data for this article were obtained through an initial Medline search and from the references of relevant articles, and used to provide updated information on the relationship between alcohol consumption and the hepatitis C virus. Results: Excessive alcohol consumption among patients with chronic hepatitis C is likely to result in more severe hepatic injuries, promote pathologic progression to cirrhosis, and increase the risk of developing hepatocellular carcinoma. Although the exact mechanisms involved in the progression of chronic hepatitis C in alcoholic patients have not been definitely established, possible alcohol-induced enhancement of viral replication, iron overload, immunologic suppression, the role of NF-kappa B, and the signaling pathways involved in its activation, have been suggested. Significant correlations have been reported between hepatitis C virus RNA levels and the amount of alcohol consumed by an individual. Interferon therapy is less effective for alcohol patients, than non-alcoholic patients, even after a period of abstinence. The obtained data suggest that a hepatitis C virus infection is an important cofactor in the pathogenesis of liver disease among patients with an alcohol problem. Conclusions: In light of a possible synergistic effect between alcohol and hepatitis C virus replication, total abstention ought to be recommended, and due to alcohol's inhibitory effect on interferon therapy, patients with alcohol problems should not be treated until they stop drinking.

Antitumor activity and immunogenicity of recombinant vaccinia virus expressing HPV 16 E7 protein SigE7LAMP is enhanced by high-level coexpression of IGFBP-3

We constructed recombinant vaccinia viruses (VACVs) coexpressing the insulin-like growth factor-binding protein-3 (IGFBP-3) gene and the fusion gene encoding the SigE7Lamp antigen. The expression of the IGFBP-3 transgene was regulated either by the early H5 promoter or by the synthetic early/late (E/L) promoter. We have shown that IGFBP-3 expression regulated by the H5 promoter yielded higher amount of IGFBP-3 protein when compared with the E/L promoter. The immunization with P13-SigE7Lamp-H5-IGFBP-3 virus was more effective in inhibiting the growth of TC-1 tumors in mice and elicited higher T-cell response against VACV-encoded antigen than the P13-SigE7Lamp-TK(-) control virus. We found that high-level production of IGFBP-3 enhanced virus replication both in vitro and in vivo, resulting in more profound antigen stimulation. Production of IGFBP-3 was associated with a higher adsorption rate of P13-SigE7Lamp-H5-IGFBP-3 to CV-1 cells when compared with P13-SigE7Lamp-TK(-). Intracellular mature virions (IMVs) of the IGFBP-3-expressing virus P13-SigE7Lamp-H5-IGFBP-3 have two structural differences: they incorporate the IGFBP-3 protein and they have elevated phosphatidylserine (PS) exposure on outer membrane that could result in increased uptake of IMVs by macropinocytosis. The IMV PS content was measured by flow cytometry using microbeads covered with immobilized purified VACV virions.

Association of a microRNA‐323b polymorphism with the persistence of hepatitis B virus infection by the enhancement of viral replication

Recent studies have shown that some mammalian microRNAs (miRNAs) play a role in antiviral defence. However, little is known about the role of miRNA-323b in hepatitis B virus (HBV)-host interaction. We explored whether single nucleotide polymorphism (SNP) of miRNA-323b affects HBV replication in a Korean HBV cohort. Genotyping was performed in a total of 1439 subjects composed of 404 spontaneously recovered (SR) subjects as normal controls and 1035 chronic carriers (CC) of HBV who were further classified into 313 patients with chronic hepatitis, 305 patients with liver cirrhosis and 417 patients with hepatocellular carcinoma. To confirm the effect of SNP of miRNA-323b on HBV replication in vitro, HepAD38 cells were transfected with miRNA-323b wild type or miRNA-323b SNP plasmid vectors, and HBV replication was induced for 5days. HBV DNA was isolated and quantified using real-time PCR. The polymorphism rs56103835C>T in the pre-miRNA region of miRNA-323b revealed significant minor allele frequency (0.273). rs56103835C>T SNP showed significantly affect persistence of HBV in CC group compared with SR group (OR=1.29, P=0.009 in a codominant model; OR = 1.29, P =0.03 in a dominant model; and OR=1.78, P=0.03 in a recessive model). In vitro, the total intracellular HBV DNA content was significantly reduced by miRNA-323b wild-type plasmid vector transfection (P=0.014). The polymorphism of miRNA-323b was significantly associated with persistence of HBV by the enhancement of HBV replication (P=0.021). Our findings provide a novel perspective on the role SNP of miRNAs in host-virus interactions in HBV infection.

HnRNP A2/B1 interacts with influenza A viral protein NS1 and inhibits virus replication potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nuclear export

The NS1 protein of influenza viruses is a major virulence factor and exerts its function through interacting with viral/cellular RNAs and proteins. In this study, we identified heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) as an interacting partner of NS1 proteins by a proteomic method. Knockdown of hnRNP A2/B1 by small interfering RNA (siRNA) resulted in higher levels of NS vRNA, NS1 mRNA, and NS1 protein in the virus-infected cells. In addition, we demonstrated that hnRNP A2/B1 proteins are associated with NS1 and NS2 mRNAs and that knockdown of hnRNP A2/B1 promotes transport of NS1 mRNA from the nucleus to the cytoplasm in the infected cells. Lastly, we showed that knockdown of hnRNP A2/B1 leads to enhanced virus replication. Our results suggest that hnRNP A2/B1 plays an inhibitory role in the replication of influenza A virus in host cells potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nucleocytoplasmic translocation.

HIV-1 infection of macrophages dysregulates innate immune responses to Mycobacterium tuberculosis by inhibition of interleukin-10.

Human immunodeficiency virus (HIV)-1 and Mycobacterium tuberculosis (M. tuberculosis) both target macrophages, which are key cells in inflammatory responses and their resolution. Therefore, we tested the hypothesis that HIV-1 may modulate macrophage responses to coinfection with M. tuberculosis. HIV-1 caused exaggerated proinflammatory responses to M. tuberculosis that supported enhanced virus replication, and were associated with deficient stimulus-specific induction of anti-inflammatory interleukin (IL)-10 and attenuation of mitogen-activated kinase signaling downstream of Toll-like receptor 2 and dectin-1 stimulation. Our in vitro data were mirrored by lower IL-10 and higher proinflammatory IL-1β in airway samples from HIV-1-infected patients with pulmonary tuberculosis compared with those with non-tuberculous respiratory tract infections. Single-round infection of macrophages with HIV-1 was sufficient to attenuate IL-10 responses, and antiretroviral treatment of replicative virus did not affect this phenotype. We propose that deficient homeostatic IL-10 responses may contribute to the immunopathogenesis of active tuberculosis and propagation of virus infection in HIV-1/M. tuberculosis coinfection.

Involvement of the Cellular Phosphatase DUSP1 in Vaccinia Virus Infection

Poxviruses encode a large variety of proteins that mimic, block or enhance host cell signaling pathways on their own benefit. It has been reported that mitogen-activated protein kinases (MAPKs) are specifically upregulated during vaccinia virus (VACV) infection. Here, we have evaluated the role of the MAPK negative regulator dual specificity phosphatase 1 (DUSP1) in the infection of VACV. We demonstrated that DUSP1 expression is enhanced upon infection with the replicative WR virus and with the attenuated VACV viruses MVA and NYVAC. This upregulation is dependent on early viral gene expression. In the absence of DUSP1 in cultured cells, there is an increased activation of its molecular targets JNK and ERK and an enhanced WR replication. Moreover, DUSP1 knock-out (KO) mice are more susceptible to WR infection as a result of enhanced virus replication in the lungs. Significantly, MVA, which is known to produce non-permissive infections in most mammalian cell lines, is able to grow in DUSP1 KO immortalized murine embryo fibroblasts (MEFs). By confocal and electron microscopy assays, we showed that in the absence of DUSP1 MVA morphogenesis is similar as in permissive cell lines and demonstrated that DUSP1 is involved at the stage of transition between IVN and MV in VACV morphogenesis. In addition, we have observed that the secretion of pro-inflammatory cytokines at early times post-infection in KO mice infected with MVA and NYVAC is increased and that the adaptive immune response is enhanced in comparison with WT-infected mice. Altogether, these findings reveal that DUSP1 is involved in the replication and host range of VACV and in the regulation of host immune responses through the modulation of MAPKs. Thus, in this study we demonstrate that DUSP1 is actively involved in the antiviral host defense mechanism against a poxvirus infection.

Vaccinia Virus-mediated Expression of Human Erythropoietin in Tumors Enhances Virotherapy and Alleviates Cancer-related Anemia in Mice

Recombinant human erythropoietin (rhEPO), a glycoprotein hormone regulating red blood cell (RBC) formation, is used for the treatment of cancer-related anemia. The effect of rhEPO on tumor growth, however, remains controversial. Here, we report the construction and characterization of the recombinant vaccinia virus (VACV) GLV-1h210, expressing hEPO. GLV-1h210 was shown to replicate in and kill A549 lung cancer cells in culture efficiently. In mice bearing A549 lung cancer xenografts, treatment with a single intravenous dose of GLV-1h210 resulted in tumor-specific production and secretion of functional hEPO, which exerted an effect on RBC progenitors and precursors in the mouse bone marrow, leading to a significant increase in the number of RBCs and in the level of hemoglobin. Furthermore, virally expressed hEPO, but not exogenously added rhEPO, enhanced virus-mediated green fluorescent protein (GFP) expression in tumors and subsequently accelerated tumor regression when compared with the treatment with the parental virus GLV-1h68 or GLV-1h209 that expressed a nonfunctional hEPO protein. Moreover, intratumorally expressed hEPO caused enlarged tumoral microvessels, likely facilitating virus spreading. Taken together, VACV-mediated intratumorally expressed hEPO not only enhanced oncolytic virotherapy but also simultaneously alleviated cancer-related anemia.

Autophagy Benefits the Replication of Newcastle Disease Virus in Chicken Cells and Tissues

Newcastle disease virus (NDV) is an important avian pathogen. We previously reported that NDV triggers autophagy in U251 glioma cells, resulting in enhanced virus replication. In this study, we investigated whether NDV triggers autophagy in chicken cells and tissues to enhance virus replication. We demonstrated that NDV infection induced steady-state autophagy in chicken-derived DF-1 cells and in primary chicken embryo fibroblast (CEF) cells, evident through increased double- or single-membrane vesicles, the accumulation of green fluorescent protein (GFP)-LC3 dots, and the conversion of LC3-I to LC3-II. In addition, we measured autophagic flux by monitoring p62/SQSTM1 degradation, LC3-II turnover, and GFP-LC3 lysosomal delivery and proteolysis, to confirm that NDV infection induced the complete autophagic process. Inhibition of autophagy by pharmacological inhibitors and RNA interference reduced virus replication, indicating an important role for autophagy in NDV infection. Furthermore, we conducted in vivo experiments and observed the conversion of LC3-I to LC3-II in heart, liver, spleen, lung, and kidney of NDV-infected chickens. Regulation of the induction of autophagy with wortmannin, chloroquine, or starvation treatment affects NDV production and pathogenesis in tissues of both lung and intestine; however, treatment with rapamycin, an autophagy inducer of mammalian cells, showed no detectable changes in chicken cells and tissues. Moreover, administration of the autophagy inhibitor wortmannin increased the survival rate of NDV-infected chickens. Our studies provide strong evidence that NDV infection induces autophagy which benefits NDV replication in chicken cells and tissues.

Oxidative Stress Enhances Neurodegeneration Markers Induced by Herpes Simplex Virus Type 1 Infection in Human Neuroblastoma Cells

Mounting evidence suggests that Herpes simplex virus type 1 (HSV-1) is involved in the pathogenesis of Alzheimer’s disease (AD). Previous work from our laboratory has shown HSV-1 infection to induce the most important pathological hallmarks of AD brains. Oxidative damage is one of the earliest events of AD and is thought to play a crucial role in the onset and development of the disease. Indeed, many studies show the biomarkers of oxidative stress to be elevated in AD brains. In the present work the combined effects of HSV-1 infection and oxidative stress on Aβ levels and autophagy (neurodegeneration markers characteristic of AD) were investigated. Oxidative stress significantly potentiated the accumulation of intracellular Aβ mediated by HSV-1 infection, and further inhibited its secretion to the extracellular medium. It also triggered the accumulation of autophagic compartments without increasing the degradation of long-lived proteins, and enhanced the inhibition of the autophagic flux induced by HSV-1. These effects of oxidative stress were not due to enhanced virus replication. Together, these results suggest that HSV-1 infection and oxidative damage interact to promote the neurodegeneration events seen in AD.

Establishment of cell lines with increased susceptibility to EV71/CA16 by stable overexpression of SCARB2

BackgroundHuman enterovirus type 71 (EV71) and Coxsackievirus A group type 16 (CA16) belong to human Enterovirus species A of the family Picornaviridae. These viruses are recognized as the major pathogens responsible for epidemics of hand-foot-mouth disease (HFMD), which presents with fever and vesicular eruptions of palms, soles of the feet or mouth. Human scavenger receptor class B, member 2 (SCARB2) has been identified as the receptor for both EV71 and CA16, as overexpression of SCARB2 in cells can enhance virus replication significantly.MethodsIn this study, we used a lentivirus packaging vector to transduce the SCARB2 gene into human embryonic kidney cells (293), human rhabdomyosarcoma cells (RD) and African green monkey kidney cells (Vero) to create stable expression lines. Expression of SCARB2 in the resulting three transgenic cell lines was confirmed by real-time RT-PCR, immunofluorescence and flow cytometry.ResultsLevels of SCARB2 mRNA determined by real-time RT-PCR in 293-SCARB2 (293S) or RD-SCARB2 (RDS) transgenic cell lines were approximately 2 × 102 times higher than those in 293 and RD cells, respectively, and three times higher in Vero-SCARB2 (VeroS) than in Vero cells. Furthermore, EV71 and CA16 virus titers in 293S and RDS cells were 102–103-fold higher (detected in RD cell) than those in the parental cells, and a 10-fold higher titer of EV71 was achieved in VeroS cells compared with that in Vero cells.ConclusionsWe established for the first time three cell lines stably overexpressing SCARB2, which showed drastic increases in susceptibility to EV71/CA16 infection. These optimal cell lines may be utilized to develop inactivated vaccines for EV71/CA16 and facilitate rapid detection and isolation of HFMD pathogens or other Enterovirus serotypes. Furthermore, these stable cell lines also can serve as tools to facilitate drug screenings as well as molecular studies on virus-host interactions and pathogenesis of causative agents for HFMD.

Immunization against Small Ruminant Lentiviruses

Multisystemic disease caused by Small Ruminant Lentiviruses (SRLV) in sheep and goats leads to production losses, to the detriment of animal health and welfare. This, together with the lack of treatments, has triggered interest in exploring different strategies of immunization to control the widely spread SRLV infection and, also, to provide a useful model for HIV vaccines. These strategies involve inactivated whole virus, subunit vaccines, DNA encoding viral proteins in the presence or absence of plasmids encoding immunological adjuvants and naturally or artificially attenuated viruses. In this review, we revisit, comprehensively, the immunization strategies against SRLV and analyze this double edged tool individually, as it may contribute to either controlling or enhancing virus replication and/or disease.

MHC Class II–Alpha Chain Knockout Mice Support Increased Viral Replication That Is Independent of Their Lack of MHC Class II Cell Surface Expression and Associated Immune Function Deficiencies

MHCII molecules are heterodimeric cell surface proteins composed of an α and β chain. These molecules are almost exclusively expressed on thymic epithelium and antigen presenting cells (APCs) and play a central role in the development and function of CD4 T cells. Various MHC-II knockout mice have been generated including MHC-IIAα-/- (I-Aα-/-), MHC-IIAβ-/- (I-β-/-) and the double knockout (I-Aαxβ-/-). Here we report a very striking observation, namely that alphaviruses including the avirulent strain of Semliki Forest virus (aSFV), which causes asymptomatic infection in wild-type C57BL6/J (B6) mice, causes a very acute and lethal infection in I-Aα-/-, but not in I-β-/- or I-Aαxβ-/-, mice. This susceptibility to aSFV is associated with high virus titres in muscle, spleen, liver, and brain compared to B6 mice. In addition, I-Aα-/- mice show intact IFN-I responses in terms of IFN-I serum levels and IFN-I receptor expression and function. Radiation bone marrow chimeras of B6 mice reconstituted with I-Aα-/- bone marrow expressed B6 phenotype, whereas radiation chimeras of I-Aα-/- mice reconstituted with B6 bone marrow expressed the phenotype of high viral susceptibility. Virus replication experiments both in vivo and in vitro showed enhanced virus growth in tissues and cell cultures derived form I-Aα-/- compared to B6 mice. This enhanced virus replication is evident for other alpha-, flavi- and poxviruses and may be of great benefit to producers of viral vaccines. In conclusion, I-Aα-/- mice exhibit a striking susceptibility to virus infections independent of their defective MHC-II expression. Detailed genetic analysis will be carried out to characterise the underlining genetic defects responsible for the observed phenomenon.

Interaction of CtBP with adenovirus E1A suppresses immortalization of primary epithelial cells and enhances virus replication during productive infection

Adenovirus E1A induces cell proliferation, oncogenic transformation and promotes viral replication through interaction with p300/CBP, TRRAP/p400 multi-protein complex and the retinoblastoma (pRb) family proteins through distinct domains in the E1A N-terminal region. The C-terminal region of E1A suppresses E1A/Ras co-transformation and interacts with FOXK1/K2, DYRK1A/1B/HAN11 and CtBP1/2 (CtBP) protein complexes. To specifically dissect the role of CtBP interaction with E1A, we engineered a mutation (DL→AS) within the CtBP-binding motif, PLDLS, and investigated the effect of the mutation on immortalization and Ras cooperative transformation of primary cells and viral replication. Our results suggest that CtBP–E1A interaction suppresses immortalization and Ras co-operative transformation of primary rodent epithelial cells without significantly influencing the tumorigenic activities of transformed cells in immunodeficient and immunocompetent animals. During productive infection, CtBP–E1A interaction enhances viral replication in human cells. Between the two CtBP family proteins, CtBP2 appears to restrict viral replication more than CtBP1 in human cells.

2-Aminopurine Enhances the Oncolytic Activity of an E1b-Deleted Adenovirus in Hepatocellular Carcinoma Cells

Adenoviruses with deletions of viral genes have been extensively studied as potential cancer therapeutics. Although a high degree of cancer selectivity has been demonstrated with these conditionally replicating adenoviruses, low levels of virus replication can be detected in normal cells. Furthermore, these mutations were also found to reduce the activity of the replicating viruses in certain cancer cells. Recent studies have shown that co-administration of chemotherapeutic drugs may increase the activity of these viruses without affecting their specificity. We constructed an adenovirus with deletions of both the E1b and the VA-RNA genes and found that replication of this virus was selective for human hepatocellular carcinoma (HCC) cell lines when compared to normal cell lines. Furthermore, we show that 2-aminopurine (2′AP) treatment selectively enhanced virus replication and virus-mediated death of HCC cells. 2′AP did not compensate for the loss of VA-RNA activities, but rather the loss of an E1b-55K activity, such as the DNA damage response, suggesting that co-administration of 2′AP derivatives that block host DNA damage response, may increase the oncolytic activity of AdΔE1bΔVA without reducing its selectivity for HCC cells.