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Jean-Yves Sgro
Inst. for Mol.Virology
731B Bock Labs
1525 Linden Drive Madison, WI 53706

Table of Contents for this page:

  • Current Issue
  • Current Issue of Virology Journal

    Virology Journal - Latest Articles

  • Influenza polymerase encoding mRNAs utilize atypical mRNA nuclear export

  • Background: Influenza is a segmented negative strand RNA virus. Each RNA segment is encapsulated by influenza nucleoprotein and bound by the viral RNA dependent RNA polymerase (RdRP) to form viral ribonucleoproteins responsible for RNA synthesis in the nucleus of the host cell. Influenza transcription results in spliced mRNAs (M2 and NS2), intron-containing mRNAs (M1 and NS1), and intron-less mRNAs (HA, NA, NP, PB1, PB2, and PA), all of which undergo nuclear export into the cytoplasm for translation. Most cellular mRNA nuclear export is Nxf1-mediated, while select mRNAs utilize Crm1. Methods: Here we inhibited Nxf1 and Crm1 nuclear export prior to infection with influenza A/Udorn/307/1972(H3H2) virus and analyzed influenza intron-less mRNAs using cellular fractionation and reverse transcription - quantitative polymerase chain reaction (RT-qPCR). We examined direct interaction between Nxf1 and influenza intron-less mRNAs using immuno purification of Nxf1 and RT-PCR of associated RNA. Results: Inhibition of Nxf1 resulted in less influenza intron-less mRNA export into the cytoplasm for HA and NA influenza mRNAs in both human embryonic kidney cell line (293 T) and human lung adenocarcinoma epithelial cell line (A549). However, in 293 T cells no change was observed for mRNAs encoding the components of the viral ribonucleoproteins; NP, PA, PB1, and PB2, while in A549 cells, only PA, PB1, and PB2 mRNAs, encoding the RdRP, remained unaffected; NP mRNA was reduced in the cytoplasm. In A549 cells NP, NA, HA, mRNAs were found associated with Nxf1 but PA, PB1, and PB2 mRNAs were not. Crm1 inhibition also resulted in no significant difference in PA, PB1, and PB2 mRNA nuclear export. Conclusions: These results further confirm Nxf1-mediated nuclear export is functional during the influenza life cycle and hijacked for select influenza mRNA nuclear export. We reveal a cell type difference for Nxf1-mediated nuclear export of influenza NP mRNA, a reminder that cell type can influence molecular mechanisms. Importantly, we conclude that in both A549 and 293 T cells, PA, PB1, and PB2 mRNA nuclear export is Nxf1 and Crm1 independent. Our data support the hypothesis that PA, PB1, and PB2 mRNAs, encoding the influenza RdRP, utilize atypical mRNA nuclear export.

  • Seroprevalence of HHV-6 and HHV-8 among blood donors in Greece

  • Background: Herpes viruses infection transmitted through healthy but infected blood donors pose a danger to herpes-naive immunocompromised recipients. The risk of transfusion-related HHV-8 transmission is different in endemic and not endemic areas. HHV-6 and HHV-8 seroprevalence and viral load among blood donors have been reported from different countries. The aim of our study was to assess the seroprevalence of HHV-8 and HHV-6 in volunteer blood donors from Greece which is unknown.FindingsSerum samples from 179 healthy blood donors were tested for the presence of IgG antibodies against HHV-6 and HHV-8 with ELISA. None of the 179 donors of Greek origin tested was positive for HHV-8. HHV-6 seropositivity was assessed in 160 blood donorsaapos; samples and was found to be 78.75% (126/160). The HHV-6 seroprevalence did not differ either between males and females or among different decade age groups. Conclusions: The fact, that no blood donor was positive for HHV-8 IgG antibodies indicates that the risk for transfusion related HHV-8 transmission in Greece, if any, is negligible and does not warrant broad testing for HHV-8. Definitely further studies are needed, in order to clarify the potential risk of HHV-6 transmission.

  • Identification of molecular sub-networks associated with cell survival in a chronically SIVmac-infected human CD4+ T cell line

  • Background: The deciphering of cellular networks to determine susceptibility to infection by HIV or the related simian immunodeficiency virus (SIV) is a major challenge in infection biology. Results: Here, we have compared gene expression profiles of a human CD4+ T cell line at 24 h after infection with a cell line of the same origin permanently releasing SIVmac. A new knowledge-based-network approach (Inter-Chain-Finder, ICF) has been used to identify sub-networks associated with cell survival of a chronically SIV-infected T cell line. Notably, the method can identify not only differentially expressed key hub genes but also non-differentially expressed, critical, aapos;hiddenaapos; regulators. Six out of the 13 predicted major hidden key regulators were among the landscape of proteins known to interact with HIV. Several sub-networks were dysregulated upon chronic infection with SIV. Most prominently, factors reported to be engaged in early stages of acute viral infection were affected, e.g. entry, integration and provirus transcription and other cellular responses such as apoptosis and proliferation were modulated. For experimental validation of the gene expression analyses and computational predictions, individual pathways/sub-networks and significantly altered key regulators were investigated further. We showed that the expression of caveolin-1 (Cav-1), the top hub in the affected protein-protein interaction network, was significantly upregulated in chronically SIV-infected CD4+ T cells. Cav-1 is the main determinant of caveolae and a central component of several signal transduction pathways. Furthermore, CD4 downregulation and modulation of the expression of alternate and co-receptors as well as pathways associated with viral integration into the genome were also observed in these cells. Putatively, these modifications interfere with re-infection and the early replication cycle and inhibit cell death provoked by syncytia formation and bystander apoptosis. Conclusions: Thus, by using the novel approach for network analysis, ICF, we predict that in the T cell line chronically infected with SIV, cellular processes that are known to be crucial for early phases of HIV /SIV replication are altered and cellular responses that result in cell death are modulated. These modifications presumably contribute to cell survival despite chronic infection.

  • Antiviral potency and functional analysis of tetherin orthologues encoded by horse and donkey

  • Background: Tetherin is an interferon-inducible host cell factor that blocks the viral particle release of the enveloped viruses. Most knowledge regarding the interaction between tetherin and viruses has been obtained using the primate lentiviral system. However, much less is known about the functional roles of tetherin on other lentiviruses. Equine infectious anemia virus (EIAV) is an important macrophage-tropic lentivirus that has been widely used as a practical model for investigating the evolution of the host-virus relationship. The host range of EIAV is reported to include all members of the Equidae family. However, EIAV has different clinical responses in horse and donkey. It’s intriguing to investigate the similarities and differences between the tetherin orthologues encoded by horse and donkey. Results: We report here that there are two equine tetherin orthologues. Compared to horse tetherin, there are three valine amino acid deletions within the transmembrane domain and three distinct mutations within the ectodomain of donkey tetherin. However, the antiviral activity of donkey tetherin was not affected by amino acid deletion or substitution. In addition, both tetherin orthologues encoded by horse and donkey are similarly sensitive to EIAV Env protein, and equally activate NF-κB signaling. Conclusion: Our data suggest that both tetherin orthologues encoded by horse and donkey showed similar antiviral activities and abilities to induce NF-κB signaling. In addition, the phenomenon about the differential responses of horses and donkeys to infection with EIAV was not related with the differences in the structure of the corresponding tetherin orthologues.

  • Substitution of the premembrane and envelope protein genes of Modoc virus with the homologous sequences of West Nile virus generates a chimeric virus that replicates in vertebrate but not mosquito cells

  • Background: Most known flaviviruses, including West Nile virus (WNV), are maintained in natural transmission cycles between hematophagous arthropods and vertebrate hosts. Other flaviviruses such as Modoc virus (MODV) and Culex flavivirus (CxFV) have host ranges restricted to vertebrates and insects, respectively. The genetic elements that modulate the differential host ranges and transmission cycles of these viruses have not been identified. Methods: Fusion polymerase chain reaction (PCR) was used to replace the capsid (C), premembrane (prM) and envelope (E) genes and the prM-E genes of a full-length MODV infectious cDNA clone with the corresponding regions of WNV and CxFV. Fusion products were directly transfected into baby hamster kidney-derived cells that stably express T7 RNA polymerase. At 4 days post-transfection, aliquots of each supernatant were inoculated onto vertebrate (BHK-21 and Vero) and mosquito (C6/36) cells which were then assayed for evidence of viral infection by reverse transcription-PCR, Western blot and plaque assay. Results: Chimeric virus was recovered in cells transfected with the fusion product containing the prM-E genes of WNV. The virus could infect vertebrate but not mosquito cells. The in vitro replication kinetics and yields of the chimeric virus were similar to MODV but the chimeric virus produced larger plaques. Chimeric virus was not recovered in cells transfected with any of the other fusion products. Conclusions: Our data indicate that genetic elements outside of the prM-E gene region of MODV condition its vertebrate-specific phenotype.

  • Chitosan microparticles loaded with yeast-derived PCV2 virus-like particles elicit antigen-specific cellular immune response in mice after oral administration

  • Background: Porcine circovirus type 2 (PCV2)-associated diseases are a major problem for the swine industry worldwide. In addition to improved management and husbandry practices, the availability of several anti-PCV2 vaccines provides an efficient immunological option for reducing the impact of these diseases. Most anti-PCV2 vaccines are marketed as injectable formulations. Although these are effective, there are problems associated with the use of injectable products, including laborious and time-consuming procedures, the induction of inflammatory responses at the injection site, and treatment-associated stress to the animals. Oral vaccines represent an improvement in antigen delivery technology; they overcome the problems associated with injection management and facilitate antigen boosting when an animals’ immunity falls outside the protective window. Methods: Chitosan microparticles were used as both a vehicle and mucosal adjuvant to deliver yeast-derived PCV2 virus-like particles (VLPs) in an attempt to develop an oral vaccine. The physical characteristics of the microparticles, including size, Zeta potential, and polydispersity, were examined along with the potential to induce PCV2-specific cellular immune responses in mice after oral delivery. Results: Feeding mice with PCV2 VLP-loaded, positively-charged chitosan microparticles with an average size of 2.5 μm induced the proliferation of PCV2-specific splenic CD4+/CD8+ lymphocytes and the subsequent production of IFN-γ to levels comparable with those induced by an injectable commercial formulation. Conclusion: Chitosan microparticles appear to be a safe, simple system on which to base PCV2 oral vaccines. Oral chitosan-mediated antigen delivery is a novel strategy that efficiently induces anti-PCV2 cellular responses in a mouse model. Further studies in swine are warranted.

  • Quantitation of substitutions at amino acid 70 in hepatitis C virus genotype 1b

  • Background: Substitutions of amino acid (aa) 70 in the core region of hepatitis C virus genotype 1b (HCV 1b) are a predictor of the non-virological response to pegylated interferon plus ribavirin (PEG-IFN/RBV) therapy. The aim of our study was to develop quantitative real-time reverse transcription polymerase chain reaction (qPCR) assays to quantify wild-type (70 W) and mutant (70 M) strains of HCV 1b. Methods: We used the TaqMan system to quantify strains 70 W and 70 M. Codon 70 in the HCV 1b core region can be either CGN or CAN, therefore degenerate TaqMan minor groove binder (MGB) probes with inosine were used. We determined detection limits, sensitivity and specificity of the methods developed. Direct sequencing and cloning of the HCV core region were used to confirm the reliability of our new system. Serum samples from 138 Chinese patients infected with HCV 1b were examined with the system we developed and compared with results obtained from the Roche TaqMan RT-PCR HCV RNA quantitation system. Results: Degenerate MGB probes were able to clearly distinguish 70 W from 70 M. The detection limit was 103 copies/mL. Cross-reactivity tests confirmed the specificity of our method. Our system can effectively quantify 70 W and 70 M for 99.6% of patients with HCV 1b. Further tests involving cloning and sequencing confirmed the reliability of our system. Conclusions: We developed an assay system using degenerate TaqMan MGB probes with inosine to quantify wild-type and mutant viral RNAs of the HCV 1b core region at aa 70. Our developed assay system had high levels of sensitivity and accuracy, and could prove useful in investigating dynamic changes during PEG-IFN/RBV therapy to assess virological responses.

  • Phylogenetic relationships and pathogenicity variation of two Newcastle disease viruses isolated from domestic ducks in Southern China

  • Background: Newcastle disease (ND) is an OIE listed disease caused by virulent avian paramyxovirus type 1 (APMV-1) strains, which is enzootic and causes large economic losses in the poultry sector. Genotype VII and genotype IX NDV viruses were the predominant circulating genotype in China, which may possibly be responsible for disease outbreaks in chicken flocks in recent years. While ducks and geese usually have exhibited inapparent infections. Methods: In the present study, we investigate the complete genome sequence, the clinicopathological characterization and transmission of two virulent Newcastle disease viruses, SS-10 and NH-10, isolated from domestic ducks in Southern China in 2010. Results: F, and the complete gene sequences based on phylogenetic analysis demonstrated that SS-10 (genotype VII) and NH-10 (genotype IX) belongs to class II. The deduced amino acid sequence was (112)R-R-Q-K/R-R-F(117) at the fusion protein cleavage site. Animal experiment results showed that the SS-10 virus isolated from ducks was highly pathogenic for chickens and geese, but low pathogenic for ducks. It could be detected from spleen, lung, kidney, trachea, small intestine, bursa of fabricius, thymus, pancreas and cecal tonsils, oropharyngeal and cloacal swabs, and could transmit to the naive contact birds. Moreover, it could transmit to chickens, ducks and geese by naive contact. However, the NH-10 virus isolated from ducks could infect some chickens, ducks and geese, but only caused chickens to die. Additionally, it could transmit to the naive contact chickens, ducks, and geese. Conclusion: The two NDV isolates exhibited different biological properties with respect to pathogenicity and transmission in chickens, ducks and geese. Therefore, no species-preference exists for chicken, duck or goose viruses and more attention should be paid to the trans-species transmission of VII NDVs between ducks, geese and chickens for the control and eradication of ND.

  • Identification and characterization of unrecognized viruses in stool samples of non-polio acute flaccid paralysis children by simplified VIDISCA

  • Background: The use of sequence independent methods combined with next generation sequencing for identification purposes in clinical samples appears promising and exciting results have been achieved to understand unexplained infections. One sequence independent method, Virus Discovery based on cDNA Amplified Fragment Length Polymorphism (VIDISCA) is capable of identifying viruses that would have remained unidentified in standard diagnostics or cell cultures. Methods: VIDISCA is normally combined with next generation sequencing, however, we set up a simplified VIDISCA which can be used in case next generation sequencing is not possible. Stool samples of 10 patients with unexplained acute flaccid paralysis showing cytopathic effect in rhabdomyosarcoma cells and/or mouse cells were used to test the efficiency of this method. To further characterize the viruses, VIDISCA-positive samples were amplified and sequenced with gene specific primers. Results: Simplified VIDISCA detected seven viruses (70%) and the proportion of eukaryotic viral sequences from each sample ranged from 8.3 to 45.8%. Human enterovirus EV-B97, EV-B100, echovirus-9 and echovirus-21, human parechovirus type-3, human astrovirus probably a type-3/5 recombinant, and tetnovirus-1 were identified. Phylogenetic analysis based on the VP1 region demonstrated that the human enteroviruses are more divergent isolates circulating in the community. Conclusion: Our data support that a simplified VIDISCA protocol can efficiently identify unrecognized viruses grown in cell culture with low cost, limited time without need of advanced technical expertise. Also complex data interpretation is avoided thus the method can be used as a powerful diagnostic tool in limited resources. Redesigning the routine diagnostics might lead to additional detection of previously undiagnosed viruses in clinical samples of patients.

  • Mouse Cytomegalovirus infection overrules T regulatory cell suppression on natural killer cells

  • Background: Cytomegalovirus establishes lifelong persistency in the host and leads to life threatening situations in immunocompromised patients. FoxP3+ T regulatory cells (Tregs) critically control and suppress innate and adaptive immune responses. However, their specific role during MCMV infection, especially pertaining to their interaction with NK cells, remains incompletely defined. Methods: To understand the contribution of Tregs on NK cell function during acute MCMV infection, we infected Treg depleted and undepleted DEREG mice with WT MCMV and examined Treg and NK cell frequency, number, activation and effector function in vivo. Results: Our results reveal an increased frequency of activated Tregs within the CD4+ T cell population shortly after MCMV infection. Specific depletion of Tregs in DEREG mice under homeostatic conditions leads to an increase in NK cell number as well as to a higher activation status of these cells as compared with non-depleted controls. Interestingly, upon infection this effect on NK cells is completely neutralized in terms of cell frequency, CD69 expression and functionality with respect to IFN-γ production. Furthermore, composition of the NK cell population with regard to Ly49H expression remains unchanged. In contrast, absence of Tregs still boosts the general T cell response upon infection to a level comparable to the enhanced activation seen in uninfected mice. CD4+ T cells especially benefit from Treg depletion exhibiting a two-fold increase of CD69+ cells 40 h and IFN-γ+ cells 7 days p.i. while, MCMV infection per se induces robust CD8+ T cell activation which is also further augmented in Treg-depleted mice. Nevertheless, the viral burden in the liver and spleen remain unaltered upon Treg ablation during the course of infection. Conclusions: Thus, MCMV infection abolishes Treg suppressing effects on NK cells whereas T cells benefit from their absence during acute infection. This study provides novel information in understanding the collaborative interaction between NK cells and Tregs during a viral infection and provides further knowledge that could be adopted in therapeutic setups to improve current treatment of organ transplant patients where modulation of Tregs is envisioned as a strategy to overcome transplant rejection.
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