Structure of the vesicular stomatitis virus N⁰-P complex

Replication of non-segmented negative-strand RNA viruses requires the continuous supply of the nucleoprotein (N) in the form of a complex with the phosphoprotein (P). Here, we present the structural characterization of a soluble, heterodimeric complex between a variant of vesicular stomatitis virus N lacking its 21 N-terminal residues (N_Δ21) and a peptide of 60 amino acids (P₆₀) encompassing the molecular recognition element (MoRE) of P that binds RNA-free N (N⁰). The complex crystallized in a decameric circular form, which was solved at 3.0 Å resolution, reveals how the MoRE folds upon binding to N and competes with RNA binding and N polymerization. Small-angle X-ray scattering experiment and NMR spectroscopy on the soluble complex confirms the binding of the MoRE and indicates that its flanking regions remain flexible in the complex. The structure of this complex also suggests a mechanism for the initiation of viral RNA synthesis.     

Is cytoskeleton involved in vesicular stomatitis virus reproduction?

CER cells infected with vesicular stomatitis virus showed a morphology similar to that observed after cytochalasin B treatment. Temperature-sensitive mutants affected in envelope protein maturation did not induce those morphological changes at a nonpermissive temperature. In addition, the cytoskeleton was not implicated in vesicular stomatitis virus reproduction.     

Neutralized vesicular stomatitis virus binds to host cells by a different “receptor”

The addition of immune serum to vesicular stomatitis virus (VSV) results in the formation of VSV/antibody complexes which are non-infectious. Despite their being neutralized, these complexes still bind to host cells and are internalized at a rate equivalent to or greater than that observed for infectious virus. However, in contrast to infectious virus, the binding and uptake of neutralized VSV is not inhibited by phosphatidylserine and is partially sensitive to trypsin treatment. These results suggest that neutralized VSV binds to a different or additional cell binding site. By altering the VSV binding site, neutralizing antibodies may interfere with the fusogenic activity of G protein.     

The N(0)-binding region of the vesicular stomatitis virus phosphoprotein is globally disordered but contains transient α-helices

The phosphoprotein (P) of vesicular stomatitis virus (VSV) interacts with nascent nucleoprotein (N), forming the N(0)-P complex that is indispensable for the correct encapsidation of newly synthesized viral RNA genome. In this complex, the N-terminal region (P(NTR)) of P prevents N from binding to cellular RNA and keeps it available for encapsidating viral RNA genomes. Here, using nuclear magnetic resonance (NMR) spectroscopy and small-angle X-ray scattering (SAXS), we show that an isolated peptide corresponding to the 60 first N-terminal residues of VSV P (P(60)) and encompassing P(NTR) has overall molecular dimensions and a dynamic behavior characteristic of a disordered protein but transiently populates conformers containing α-helices. The modeling of P(60) as a conformational ensemble by the ensemble optimization method using SAXS data correctly reproduces the α-helical content detected by NMR spectroscopy and suggests the coexistence of subensembles of different compactness. The populations and overall dimensions of these subensembles are affected by the addition of stabilizing (1M trimethylamine-N-oxide) or destabilizing (6M guanidinium chloride) cosolvents. Our results are interpreted in the context of a scenario whereby VSV P(NTR) constitutes a molecular recognition element undergoing a disorder-to-order transition upon binding to its partner when forming the N(0)-P complex.     

Founder virus population related to route of virus transmission: a determinant of intrahost human immunodeficiency virus type 1 evolution?

We and others have shown that in individual human immunodeficiency virus type 1 (HIV-1) infection, the adaptive evolution of HIV-1 is influenced by host immune competence. In this study, we tested the hypothesis that in addition to selective forces operating within the host, transmission bottlenecks have an impact on HIV-1 intrahost evolution. Therefore, we studied the intrahost evolution of the V3 region of the external glycoprotein gp120 of HIV-1 during the 3- and 5-year periods following seroconversion after parenteral versus sexual (male-to-male) transmission in 41 participants of the Amsterdam prospective cohorts of homosexual men (n = 31) and intravenous drug users (IVDUs; n = 10) who were AIDS free and had comparable numbers of CD4+ cells. We observed that HIV-1 strains in homosexual men accumulated over 5 years more nonsynonymous substitutions within the V3 loop than HIV-1 strains in IVDUs as a result of lower rates of nonsynonymous evolution in both the initial 3-year period from seroconversion and the following 2-year period as well as a larger proportion of nonsynonymous back substitutions in IVDUs. The mean numbers of synonymous substitutions did not differ between the two risk groups. Since HIV-1 strains in IVDUs could be distinguished from the viruses of homosexual men based on several nucleotide substitutions of which the most conserved is a synonymous substitution at the tip of the V3 loop (GGC pattern), we studied whether the founder virus population itself has an impact on the intrahost evolution of HIV-1. The mean number of nonsynonymous substitutions accumulated over 5 years within the V3 loop was lower in 10 IVDUs infected by the HIV-1 strains with the GGC signature than in 4 IVDUs infected by HIV-1 strains lacking this pattern, while the mean numbers of synonymous substitutions were similar in the two groups.     

Limits in virus filtration capability? Impact of virus quality and spike level on virus removal with xenotropic murine leukemia virus

Virus filtration (VF) is a key step in an overall viral clearance process since it has been demonstrated to effectively clear a wide range of mammalian viruses with a log reduction value (LRV) > 4. The potential to achieve higher LRV from virus retentive filters has historically been examined using bacteriophage surrogates, which commonly demonstrated a potential of > 9 LRV when using high titer spikes (e.g. 10¹⁰ PFU/mL). However, as the filter loading increases, one typically experiences significant decreases in performance and LRV. The 9 LRV value is markedly higher than the current expected range of 4‐5 LRV when utilizing mammalian retroviruses on virus removal filters (Miesegaes et al., Dev Biol (Basel) 2010;133:3‐101). Recent values have been reported in the literature (Stuckey et al., Biotech Progr 2014;30:79‐85) of LRV in excess of 6 for PPV and XMuLV although this result appears to be atypical. LRV for VF with therapeutic proteins could be limited by several factors including process limits (flux decay, load matrix), virus spike level and the analytical methods used for virus detection (i.e. the Limits of Quantitation), as well as the virus spike quality. Research was conducted using the Xenotropic‐Murine Leukemia Virus (XMuLV) for its direct relevance to the most commonly cited document, the International Conference of Harmonization (ICH) Q5A (International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, Geneva, Switzerland, 1999) for viral safety evaluations. A unique aspect of this work is the independent evaluation of the impact of retrovirus quality and virus spike level on VF performance and LRV. The VF studies used XMuLV preparations purified by either ultracentrifugation (Ultra 1) or by chromatographic processes that yielded a more highly purified virus stock (Ultra 2). Two monoclonal antibodies (Mabs) with markedly different filtration characteristics and with similar levels of aggregate (<1.5%) were evaluated with the Ultra 1 and Ultra 2 virus preparations utilizing the Planova 20 N, a small virus removal filter. Impurities in the virus preparation ultimately limited filter loading as measured by determining the volumetric loading condition where 75% flux decay is observed versus initial conditions (V₇₅). This observation occurred with both Mabs with the difference in virus purity more pronounced when very high spike levels were used (>5 vol/vol %). Significant differences were seen for the process performance over a number of lots of the less‐pure Ultra 1 virus preparations. Experiments utilizing a developmental lot of the chromatographic purified XMuLV (Ultra 2 Development lot) that had elevated levels of host cell residuals (vs. the final Ultra 2 preparations) suggest that these contaminant residuals can impact virus filter fouling, even if the virus prep is essentially monodisperse. Process studies utilizing an Ultra 2 virus with substantially less host cell residuals and highly monodispersed virus particles demonstrated superior performance and an LRV in excess of 7.7 log₁₀. A model was constructed demonstrating the linear dependence of filtration flux versus filter loading which can be used to predict the V₇₅ for a range of virus spike levels conditions using this highly purified virus. Fine tuning the virus spike level with this model can ultimately maximize the LRV for the virus filter step, essentially adding the LRV equivalent of another process step (i.e. protein A or CEX chromatography). © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:135–144, 2015     

Construction and immune efficacy of recombinant pseudorabies virus expressing PrM-E proteins of Japanese encephalitis virus genotype І

Background

Japanese encephalitis (JE) is an arboviral disease with high case fatality rates and neurologic or psychiatric sequelae among survivors in Asia, western Pacific countries and northern Australia. Japanese encephalitis virus (JEV) is the cause of JE and the emergence of genotype ? (GI) JEV has displaced genotype III (GIII) as the dominant strains circulating in some Asian regions. The currently available JE vaccines are safe and effective in preventing this disease, but they are developed based on the GIII JEV strains.

Methods

The recombinant virus PRV TK^?/gE^?/PrM-E⁺ which expressed the premembrane (prM) and envelope (E) proteins of JEV SX09S-01 strain (genotype I, GI) was constructed by homologous recombination between the genome of PRV TK^?/gE^?/LacZ⁺ digested with EcoRI and plasmid pIE-CAG-PrM-E-BGH. Expression of JEV PrM and E proteins was analyzed by Western blot analysis. Immune efficacy of PRV TK^?/gE^?/PrM-E⁺ was further evaluated in mouse model.

Results

A recombinant pseudorabies virus (PRV TK^?/gE^?/PrM-E⁺) was successfully constructed. Mice experiments showed that PRV TK^?/gE^?/PrM-E⁺ could induce a high level of ELISA antibodies against PRV and JEV, as well as high titer of PRV neutralizing antibodies. After challenge with 1?×?10⁷ PFU virulent JEV SX09S-01 strain, the time of death was delayed and the survival rate was improved in PRV TK^?/gE^?/PrM-E⁺ vaccinated mice.

Conclusions

PRV TK^?/gE^?/PrM-E⁺ is a potential vaccine candidate against PRV and JEV GI infection in the future.

     

Isolation of recombinant field strains of Marek’s disease virus integrated with reticuloendotheliosis virus genome fragments

Two Marek's disease virus (MDV) field strains were isolated from chickens with tumors independently from Guangdong and Guangxi provinces, and it was confirmed that there were no co-infections with reticuloendotheliosis viruses (REV) in chicken embryo fibroblast cells (CEF) in indirect fluorescence antibody test (IFA) with REV-specific monoclonal antibodies. By dot blot hybridization and PCR of genomic DNA of MDV-infected CEF, it was indicated that LTR fragments of REV genome were integrated into genome of these two MDV field strains. To amplify and clone the integrated REV LTR with MDV sequence at the junction, 4 primers from REV LTR and 7 primers from MDV genome fragment with REV LTR insertion hot points were synthesized and 28 (4x7) pairs of primers (one from REV and another from MDV for each pair) were used in PCR while using the genomic DNA of both strains as the templates. The sequence data demonstrated that both recombinant field strains contained the same REV LTR inserted into MDV at the identical sites in US fragment of the genomes. From the above, it was speculated that both recombinant field MDVs were originated from a same recombinant virus and spread among chicken flocks in two provinces.     

Discovery of the first virus, the tobacco mosaic virus: 1892 or 1898?

Two scientists contributed to the discovery of the first virus, Tobacco mosaic virus. Ivanoski reported in 1892 that extracts from infected leaves were still infectious after filtration through a Chamberland filter-candle. Bacteria are retained by such filters, a new world was discovered: filterable pathogens. However, Ivanovski probably did not grasp the full meaning of his discovery. Beijerinck, in 1898, was the first to call 'virus', the incitant of the tobacco mosaic. He showed that the incitant was able to migrate in an agar gel, therefore being an infectious soluble agent, or a 'contagium vivum fluidum' and definitively not a 'contagium fixum' as would be a bacteria. Ivanovski and Beijerinck brought unequal but decisive and complementary contributions to the discovery of viruses. Since then, discoveries made on Tobacco mosaic virus have stood out as milestones of virology history.     

Complete genome sequence of a novel hantavirus variant of Rio Mamoré virus, Maripa virus, from French Guiana

We report the first complete genome sequence of Maripa virus identified in 2009 from a patient with hantavirus pulmonary syndrome in French Guiana. Maripa virus corresponds to a new variant of the Rio Mamoré virus species in the Bunyaviridae family, genus Hantavirus.     

Epstein-Barr virus–specific cytokine-induced killer cells for treatment of Epstein-Barr virus–related malignant lymphoma

Background

Prolonged immunosuppression or delayed T-cell recovery may favor Epstein-Barr virus (EBV) infection or reactivation after allogeneic hematopoietic stem cell transplantation (HSCT), which can lead to post-transplant lymphoproliferative disease (PTLD) and high-grade malignant B-cell lymphoma. Cytokine-induced killer (CIK) cells with dual specific anti-tumor and virus-specific cellular immunity may be applied in this context.

Nuclear localization of Sindbis virus nonstructural protein nsP2

In early infection, approximately 10% of nonstructural protein nsP2 of Sindbis virus was transported into the nuclei of virus-infected BHK-21 cells. Nuclear asP2 was dominantly associated with nuclear matrix. During the course of infection, increasing amounts of nsP2 accumulated in the nuclear fraction. A prominent accumulation of nuclear nsP2 occurred early in infection, from 1 h to 3 h postinfection. Meanwhile. a weak NTPase activity was found to be associated with the immunocomplexed nsP2. Nuclear localization of nsP2 and its possible role were diseussed in relation to the inhibition of host macromolecular synthesis.     

Phylogenetic assessment of filoviruses: how many lineages of Marburg virus?

Filoviruses have to date been considered as consisting of one diverse genus (Ebola viruses) and one undifferentiated genus (Marburg virus). We reconsider this idea by means of detailed phylogenetic analyses of sequence data available for the Filoviridae: using coalescent simulations, we ascertain that two Marburg isolates (termed the "RAVN" strain) represent a quite-distinct lineage that should be considered in studies of biogeography and host associations, and may merit recognition at the level of species. In contrast, filovirus isolates recently obtained from bat tissues are not distinct from previously known strains, and should be considered as drawn from the same population. Implications for understanding the transmission geography and host associations of these viruses are discussed.     

Molecular detection of Epstein–Barr virus in different types of lymphoma

Molecular Biology Reports - Epstein-Barr virus (EBV) is a member of the γ herpesvirus subfamily. It is widely spread, potentially oncogenic and has been studied in different human cancers such...     

Comparison of the geographical distribution of feline immunodeficiency virus and feline leukemia virus infections in the United States of America (2000–2011)

Background

Although feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) have similar risk factors and control measures, infection rates have been speculated to vary in geographic distribution over North America. Since both infections are endemic in North America, it was assumed as a working hypothesis that their geographic distributions were similar. Hence, the purpose of this exploratory analysis was to investigate the comparative geographical distribution of both viral infections. Counts of FIV (n=17,108) and FeLV (n=30,017) positive serology results (FIV antibody and FeLV ELISA) were obtained for 48 contiguous states and District of Columbia of the United States of America (US) from the IDEXX Laboratories website. The proportional morbidity ratio of FIV to FeLV infection was estimated for each administrative region and its geographic distribution pattern was visualized by a choropleth map. Statistical evidence of an excess in the proportional morbidity ratio from unity was assessed using the spatial scan test under the normal probability model.

Results

This study revealed distinct spatial distribution patterns in the proportional morbidity ratio suggesting the presence of one or more relevant and geographically varying risk factors. The disease map indicates that there is a higher prevalence of FIV infections in the southern and eastern US compared to FeLV. In contrast, FeLV infections were observed to be more frequent in the western US compared to FIV. The respective excess in proportional morbidity ratio was significant with respect to the spatial scan test (p < 0.05).

Conclusions

The observed variability in the geographical distribution of the proportional morbidity ratio of FIV to FeLV may be related to the presence of an additional or unique, but yet unknown, spatial risk factor. Putative factors may be geographic variations in specific virus strains and rate of vaccination. Knowledge of these factors and the geographical distributions of these infections can inform recommendations for testing, management and prevention. However, further studies are required to investigate the potential association of these factors with FIV and FeLV.