Long-term Sudan virus Ebola survivors maintain multiple antiviral defense mechanisms.

BACKGROUND: The critical issues of sustained memory immunity following ebolavirus disease among long-term survivors (EVD) are still unclear. METHODS: Here, we examine virus-specific immune and inflammatory responses in 12 Sudan virus (SUDV) long-term survivors from Uganda's 2000-1 Gulu outbreak, 15 years after recovery following in vitro challenge. Total RNA from isolated SUDV-stimulated and unstimulated PBMCs was extracted and analyzed. Matched serum samples were also collected to determine SUDV IgG levels and functionality. RESULTS: We detected persistent humoral (58%, 7 of 12) and cellular (33%, 4 of 12) immune responses in SUDV long-term survivors and identified critical molecular mechanisms of innate and adaptive immunity. Gene expression in immune pathways, the IFN signaling system, antiviral defense response, and activation and regulation of T- and B-cell responses were observed. SUDV long-term survivors also maintained robust virus-specific IgG antibodies capable of polyfunctional responses, including neutralizing and innate Fc effector functions. CONCLUSIONS: Data integration identified significant correlations among humoral and cellular immune responses and pinpointed a specific innate and adaptive gene expression signature associated with long-lasting immunity. This could help identify natural and vaccine correlates of protection against ebolavirus disease.

Multiple viral proteins and immune response pathways act to generate robust long-term immunity in Sudan virus survivors.

BACKGROUND: Profiles of immunity developed in filovirus patients and survivors have begun to shed light on antigen-specific cellular immune responses that had been previously under-studied. However, our knowledge of the breadth and length of those responses and the viral targets which mediate long-term memory immunity still lags significantly behind. METHODS: We characterized antigen-specific immune responses in whole blood samples of fifteen years post-infected survivors of the Sudan virus (SUDV) outbreak in Gulu, Uganda (2000-2001). We examined T cell and IgG responses against SUDV complete antigen and four SUDV proteins; glycoprotein (GP), nucleoprotein (NP), and viral protein 30 (VP30), and 40 (VP40). FINDINGS: We found survivors-maintained antigen-specific CD4+ T cell memory immune responses mediated mainly by the viral protein NP. In contrast, activated CD8+ T cell responses were nearly absent in SUDV survivors, regardless of the stimulating antigen used. Analysis of anti-viral humoral immunity revealed antigen-specific IgG antibodies against SUDV and SUDV proteins. Survivor IgGs mediated live SUDV neutralization in vitro and FcγRI and FcγRIII antibody Fc-dependent responses, mainly via antibodies to the viral proteins GP and VP40. INTERPRETATION: We highlight the key role of several proteins, i.e., GP, NP, and VP40, to act as mediators of distinctive and sustained cellular memory immune responses in long-term SUDV survivors. We suggest that the inclusion of these viral proteins in vaccine development may best mimic survivor native memory immune responses with the potential of protecting against viral infection. FUNDS: This research was funded by the Defense Threat Reduction Agency (CB4088) and by the National Institute Of Allergy And Infectious Diseases of the National Institutes of Health under Award Number R01AI111516. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

The MVMp P4 promoter is a host cell-type range determinant in vivo.

The protoparvovirus early promoters, e.g. P4 of Minute Virus of Mice (MVM), play a critical role during infection. Initial P4 activity depends on the host transcription machinery only. Since this is cell-type dependent, it is hypothesized that P4 is a host cell-type range determinant. Yet host range determinants have mapped mostly to capsid, never P4. Here we test the hypothesis using the mouse embryo as a model system. Disruption of the CRE element of P4 drastically decreased infection levels without altering range. However, when we swapped promoter elements of MVM P4 with those from equivalent regions of the closely related H1 virus, we observed elimination of infection in fibroblasts and chondrocytes and the acquisition of infection in skeletal muscle. We conclude that P4 is a host range determinant and a target for modifying the productive infection potential of the virus - an important consideration in adapting these viruses for oncotherapy.

Induction of an embryonic mouse innate immune response following inoculation in utero with minute virus of mice.

UNLABELLED: We used an embryonic-infection model system to show that MVMp, the prototypic minute virus of mice (MVM) serotype and a member of the genus Protoparvovirus, triggers a comprehensive innate immune response in the developing mouse embryo. Direct inoculation of the midtrimester embryo in utero with MVMp results in a widespread, productive infection. During a 96-h infection course, embryonic beta interferon (IFN-ß) and IFN-γ transcription were induced 90- and 60-fold, respectively. IFN-ß levels correlated with the embryo viral burden, while IFN-γ levels first increased and then decreased. Production of proinflammatory cytokines, interleukin 1ß (IL-1ß) and tumor necrosis factor alpha (TNF-α), also increased, but by smaller amounts, approximately 7-fold each. We observed increased levels of downstream antiviral effector molecules, PKR and phosphorylated STAT2. Finally, we showed that there is an immune cell response to the virus infection. Infected tissues in the embryo exhibited an increased density of mature leukocytes compared to the same tissues in uninfected embryos. The responses we observed were almost completely restricted to the infected embryos. Uninfected littermates routinely exhibited small increases in innate immune components that rarely reached statistical significance compared to negative controls. Similarly, the placentae of infected embryos did not show any significant increase in transcription of innate immune cytokines. Since the placenta has both embryonic and maternal components, we suggest there is minimal involvement of the dam in the response to infection. IMPORTANCE: Interaction between the small single-stranded vertebrate DNA viruses, the protoparvoviruses, and the host innate immune system has been unclear. The issue is important practically given the potential use of these viruses as oncotherapeutic agents. The data reported here stand in contrast to studies of innate immune response during protoparvovirus infection of adult hosts, which invariably reported no or minimal and sporadic induction of an interferon response during infection. We conclude that under conditions of robust and productive MVM infection, a normal murine host is able to mount a significant and broad innate immune response.

The Minute Virus of Mice NS2 proteins are not essential for productive infection of embryonic murine cells in utero.

The P4 promoter of the autonomous parvovirus Minute Virus of Mice (MVM) drives the production of its non-structural proteins, NS1 and NS2. The NS2 isoforms are without enzymatic activity but interact with cellular proteins. While NS2 is crucial to the viral life cycle in cultured murine cells, NS2-null mutant virus productively infects transformed host cells of other species. In the mouse, sensitivity to MVM infection is age dependent, exhibiting limited subclinical infections in adults, but sustained and potentially lethal infection in embryos. We therefore questioned whether the species-dependent requirement for NS2 function in vitro would be retained in utero. We report here that it is not. NS2-null mutant MVMp is capable of mounting a productive, albeit much reduced, infection of normal embryonic mouse cells in vivo. Based on the data, we hypothesize that NS2 may bear an as-yet undescribed immunosuppressive function.

Activation of an antiviral response in normal but not transformed mouse cells: a new determinant of minute virus of mice oncotropism.

Parvovirus minute virus of mice (MVMp) is endowed with oncotropic properties so far ascribed only to the dependency of the virus life cycle on cellular factors expressed during S phase and/or modulated by malignant transformation. For other viruses oncotropism relies on their inability to circumvent type I interferon (IFN)-induced innate antiviral mechanisms, the first line of defense triggered by normal cells against viral infections. These agents propagate, therefore, preferentially in transformed/tumor cells, which often lack functional antiviral mechanisms. The present study aimed at investigating whether antiviral processes also contribute to MVMp oncotropism. Our results demonstrate that in contrast to MVMp-permissive transformed mouse A9 fibroblasts, freshly isolated normal counterparts (mouse embryonic fibroblasts [MEFs]) mount, through production and release of type I IFNs upon their infection, an antiviral response against MVMp lytic multiplication. Pretreatment of MEFs with a type I IFN-beta-neutralizing antibody, prior to MVMp infection, inhibits the virus-triggered antiviral response and improves the fulfillment of the MVMp life cycle. Our results also show that part of the A9 permissiveness to MVMp relies on the inability to produce type I IFNs upon parvovirus infection, a feature related either to an A9 intrinsic deficiency of this process or to an MVMp-triggered inhibitory mechanism, since stimulation of these cells by exogenous IFN-beta strongly inhibits the parvovirus life cycle. Taken together, our results demonstrate for the first time that parvovirus infection triggers an innate antiviral response in normal cells and suggest that the MVMp oncotropism depends at least in part on the failure of infected transformed cells to mount such a response.

An extension of the Minute Virus of Mice tissue tropism.

Well-defined tissue tropism makes Autonomous Parvoviruses a valuable model for studies of virus-cell interactions and gene therapy research. We developed a new Minute Virus of Mice variant, different from the known prototype (MVMp) and immunosuppressive (MVMi) strains. The new virus variant, designated F1, was isolated from the culture of semi-permissive Fisher Rat Fibroblasts, F111, infected with MVMp. The F1 genome carried point mutations in regions known to determine the mutually restricted host ranges of MVMp and MVMi. In F111 cells, F1 cytotoxicity, gene expression and multiplication were significantly higher compared to MVMp. Conversely the wild-type virus propagated in MVMp-permissive cells more efficiently than the F1. Reversion of the F1-specific mutations to wild-type MVMp sequence, following reverse-passaging of the mutant virus in MVMp-permissive cells, confirmed a specific adaptation of the F1 virus to F111 cells. Considerable divergence in tissue specificities between the wild-type and mutant viruses was demonstrated in vivo.

Virus-induced alterations of membrane lipids affect the incorporation of PrP Sc into cells.

Prion diseases are fatal neurodegenerative disorders characterized by long incubation periods. To investigate whether concurrent diseases can modify the clinical outcome of prion-affected subjects, we tested the effect of viral infection on the binding and internalization of PrP(Sc), essential steps of prion propagation. To this effect, we added scrapie brain homogenate or purified PrP(Sc) to fibroblasts previously infected with minute virus of mice (MVM), a mouse parvovirus. We show here that the rate of incorporation of PrP(Sc) into MVM-infected cells was significantly higher than that observed for naïve cells. Immunostaining of cells and immunoblotting of subcellular fractions using antibodies recognizing PrP and LysoTracker, a lysosomal marker, revealed that in both control and MVM-infected cells the incorporated PrP(Sc) was associated mostly with lysosomes. Interestingly, flotation gradient analysis revealed that the majority of the PrP(Sc) internalized into MVM-infected cells shifted toward raft-containing low-density fractions. Concomitantly, the MVM-infected cells demonstrated increased levels of the glycosphingolipid GM1 (an essential raft lipid component) throughout the gradient and a shift in caveolin 1 (a raft protein marker) toward lighter membrane fractions compared with noninfected cells. Our results suggest that the effect of viral infection on membrane lipid composition may promote the incorporation of exogenous PrP(Sc) into rafts. Importantly, membrane rafts are believed to be the conversion site of PrP(C) to PrP(Sc); therefore, the association of exogenous PrP(Sc) with such membrane microdomains may facilitate prion infection.

A method for the construction of equalized directional cDNA libraries from hydrolyzed total RNA.

BACKGROUND: The transcribed sequences of a cell, the transcriptome, represent the trans-acting fraction of the genetic information, yet eukaryotic cDNA libraries are typically made from only the poly-adenylated fraction. The non-coding or translated but non-polyadenylated RNAs are therefore not represented. The goal of this study was to develop a method that would more completely represent the transcriptome in a useful format, avoiding over-representation of some of the abundant, but low-complexity non-translated transcripts. RESULTS: We developed a combination of self-subtraction and directional cloning procedures for this purpose. Libraries were prepared from partially degraded (hydrolyzed) total RNA from three different species. A restriction endonuclease site was added to the 3' end during first-strand synthesis using a directional random-priming technique. The abundant non-polyadenylated rRNA and tRNA sequences were largely removed by using self-subtraction to equalize the representation of the various RNA species. Sequencing random clones from the libraries showed that 87% of clones were in the forward orientation with respect to known or predicted transcripts. 70% matched identified or predicted translated RNAs in the sequence databases. Abundant mRNAs were less frequent in the self-subtracted libraries compared to a non-subtracted mRNA library. 3% of the sequences were from known or hypothesized ncRNA loci, including five matches to miRNA loci. CONCLUSION: We describe a simple method for making high-quality, directional, random-primed, cDNA libraries from small amounts of degraded total RNA. This technique is advantageous in situations where a cDNA library with complete but equalized representation of transcribed sequences, whether polyadenylated or not, is desired.

A wide-range, low-cost 150 bp ladder for sizing DNA fragments between 150 and 4500 bp.

Agarose gel electrophoresis, a very routine procedure, requires molecular weight standards; these are usually manufactured from plasmid or viral DNA fragments, or more recently, from PCR products of defined sizes. We describe here the preparation of a molecular weight standard from a completely different DNA source - the uniquely organized genome of the beetle Tenebrio molitor. The standard can be used to accurately size DNAs between 150 and 4500 bp, a useful range of sizes for many agarose gel electrophoresis applications, including separation of PCR products and plasmid cloning targets. In addition, it is easy to prepare, inexpensive, and rivals the best of the commercial ladders.

Complete sequence of Litopenaeus vannamei (Crustacea: Decapoda) vitellogenin cDNA and its expression in endocrinologically induced sub-adult females.

The gene that encodes vitellogenin (Vg), the precursor of the major yolk protein, vitellin, is expressed during vitellogenesis in decapod crustaceans. In this study, we sequenced the full-length cDNA from the Pacific white shrimp Litopenaeus vannamei Vg gene (LvVg). This is the first open thelycum penaeid shrimp Vg cDNA to be sequenced. The transcript encodes a 2587 amino acid polypeptide with up to 85% identity to Vg of different penaeid species. Peptide mass fingerprints (PMFs) of the vitelline polypeptides suggest that the predicted endoprotease cleavage site at amino acids 725-728 does indeed undergo cleavage. Five prominent high-density lipoprotein polypeptides of masses 179, 113, 78, 61, and 42kDa were isolated from vitellogenic ovary, and their PMFs were determined by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) spectrometry. It is likely that these polypeptides are all products of the LvVg gene. Removal of the X-organ sinus gland complex (XO-SG), which secretes the neurohormones that control the endocrine system regulating molt and reproduction, can induce both these processes. During the course of a number of molt cycles in induced sub-adult females, periodic ovarian growth and resorption were observed. Ovary growth correlated with LvVg expression in both the hepatopancreas and the ovary. Expression in ovaries of induced intermolt-early premolt females was significantly higher compared to all other sub-groups. Expression in ovaries of induced females was significantly higher compared to hepatopancreas at all molt cycle stages. Periodicity of molt and vitellogenesis in endocrinologically induced sub-adult shrimps may serve as a model to study alternate regulation of gene expression during these two processes.

Expression of the reproductive female-specific vitellogenin gene in endocrinologically induced male and intersex Cherax quadricarinatus crayfish.

In oviparous females, the synthesis of the yolk precursor vitellogenin is an important step in ovarian maturation and oocyte development. In decapod Crustacea, including the red-claw crayfish (Cherax quadricarinatus), this reproductive process is regulated by inhibitory neurohormones secreted by the endocrine X-organ-sinus gland (XO-SG) complex. In males, the C. quadricarinatus vitellogenin gene (CqVg), although present, is not expressed under normal conditions. We show here that endocrine manipulation by removal of the XO-SG complex from male animals induced CqVg transcription. The CqVg gene was expressed differentially during the molt cycle in these induced males: no expression was seen in the intermolt stages, but expression was occasionally detected in the premolt stages and always detected in the early postmolt stages. Relative quantitation with a real-time reverse transcriptase-polymerase chain reaction showed that expression of CqVg in induced early postmolt males was an order of magnitude lower than that in reproductive females, a finding that was consistent with RNA in situ hybridization results. The SDS-PAGE of high-density lipoproteins from the hemolymph of endocrinologically induced early postmolt males did not show the typical vitellogenin-related polypeptide profile found in reproductive females. On the other hand, removal of the XO-SG complex from intersex individuals, which are chromosomally female but functionally male and possess an arrested female reproductive system, induced the expression, translation, and release of CqVg products into the hemolymph, as was the case for vitellogenic females. The expression of CqVg in endocrinologically manipulated molting males and intersex animals provides an inducible model for the investigation and understanding of the endocrine regulation of CqVg expression and translation in Crustacea as well as the relationship between the endocrine axes regulating molt and reproduction.

Host cell specificity of minute virus of mice in the developing mouse embryo.

Productive infection by the murine autonomous parvovirus minute virus of mice (MVM) depends on a dividing cell population and its differentiation state. We have extended the in vivo analysis of the MVM host cell type range into the developing embryo by in utero inoculation followed by further gestation. The fibrotropic p strain (MVMp) and the lymphotropic i strain (MVMi) did not productively infect the early mouse embryo but were able to infect overlapping sets of cell types in the mid- or late-gestation embryo. Both MVMp and MVMi infected developing bone primordia, notochord, central nervous system, and dorsal root ganglia. MVMp exhibited extensive infection in fibroblasts, in the epithelia of lung and developing nose, and, to a lesser extent, in the gut. MVMi also infected endothelium. The data indicated that the host ranges of the two MVM strains consist of overlapping sets of cell types that are broader than previously known from neonate and in vitro infection experiments. The correlation between MVM host cell types and the cell types that activate the transgenic P4 promoter is consistent with the hypothesis that activation of the incoming viral P4 promoter by the host cell is one of the host range determinants of MVM.

Viral inoculation of mouse embryos in utero.

A technique is described for the injection of live virus into early- and mid-gestation mouse embryos in utero. The procedure is quick, easy, harmless to the embryos, and does not require specialized surgical or microinjection equipment. Since the developing embryo contains most different cell types in a very wide range of differentiation states, the procedure permits a rapid and near complete characterization of the host cell type range in a single experimental system. Under anaesthesia, a simple laparotomy was used to reveal the uterine horns of 9.5 or 12.5 days post-conception(dpc) females. One uterine horn was deflected onto the ventral abdominal surface. Embryos were injected through the uterine wall and the uterine horn replaced into the abdominal cavity. The entire operation could be completed in 10-15 min without distinguishable pain to the mother or adverse effect on the pregnancy. The procedure is presented in sufficient detail to permit its ready adoption in situations where a more complete characterization of host cell type range is sought.

A replacement for methoxyflurane (Metofane) in open-circuit anaesthesia.

Methoxyflurane (Metofane) has been widely used as an open-circuit anaesthetic in small laboratory animals for several decades. Its low vapour pressure and high blood solubility have permitted its use in convenient and simple drop-chamber/nose-cone setups. Recently, following the decision by the primary manufacturer to discontinue production, it has become increasingly difficult to obtain methoxyflurane. We describe here a simple and effective adaptation of isoflurane, an excellent inhalation anaesthetic, to open-circuit drop-chamber/nose-cone anaesthesia. It was found that the vapour concentration of isoflurane could be continuously varied by dissolving the anaesthetic in propylene glycol and that a 20% solution produced effective anaesthesia such that in adult mice, 2 ml of 20% isoflurane in propylene glycol induced anaesthesia within 2 min in a one-litre drop chamber. Furthermore, anaesthesia maintenance with 20% isoflurane was tested in two sets of mice. In one set, surgical plane anaesthesia was maintained for 10 min in a head chamber. After removal of the chamber, the animals awoke within one minute and recovered without any indication of post-anaesthetic distress. The second set contained pregnant mice; here anaesthesia was maintained for between 10 and 12 min, during which laparotomy, exposure of one uterine horn, intrauterine injection and wound closure were completed. The recovery from anaesthesia was also within a minute and with no signs of distress. Healthy litters were delivered after a normal gestation. This isoflurane/propylene glycol procedure is simple, effective and humane, and is a good substitute for methoxyflurane.

The P4 promoter of the parvovirus minute virus of mice is developmentally regulated in transgenic P4-LacZ mice.

Activation of the minute virus of mice (MVM) P4 promoter is a key step in the life cycle of the virus and is completely dependent on host transcription factors. Since transcription-factor composition varies widely in different cell types, there is the possibility that only some cell types in the host organism have the capacity to initiate expression from the P4 promoter and therefore that the promoter may be a factor in determining the tropism of MVM. In this study, the ability of various cell types to activate P4, independent of the other virus-host interactions, was examined in transgenic mouse lines bearing a beta-galactosidase reporter sequence driven by the P4 promoter. It was found that lacZ was expressed during embryogenesis and in the adult in a cell-type-specific and differentiation-dependent pattern. The data are consistent with cell-type and stage-specific activation of the P4 promoter having a role in determining the host cell-type range of MVM. The ability of some parvoviruses to replicate in, and kill oncogenically transformed cells, and to destroy induced tumors in laboratory animals is the basis of recent approaches to use MVM-based vectors in cancer gene therapy. Since these vectors rely on the activation of the P4 promoter by the target tissues, understanding the promoter dependence on cell-type and differentiation status is important for their design and potential use.

The vitellogenin cDNA of Cherax quadricarinatus encodes a lipoprotein with calcium binding ability, and its expression is induced following the removal of the androgenic gland in a sexually plastic system.

Oocyte maturation in decapod crustaceans is a two step process. Primary vitellogenesis is followed by a variable hiatus that lasts up to the onset of secondary vitellogenesis, which is marked by the rapid accumulation of yolk proteins in the oocytes. We have cloned a complete Cherax quadricarinatus vitellogenin cDNA. The sequenced cDNA contains a 2584 aa open reading frame which shows sequence similarity to vitellogenins from other crustaceans. The mRNA encodes at least two of the previously identified vitellin components, indicating that the primary translation product is subject to post-translational modification, including proteolytic cleavage. The region close to the 3(') end of the mRNA encodes a previously characterized negatively charged protein (provisionally designated P(106)). We show here that the negative charge of P(106) could be due to its ability to bind calcium. Northern blot data show that this gene is expressed as a single 8000 nt transcript and is present in the hepatopancreas of secondary-vitellogenic females. Primary vitellogenic and other tissues examined in male and female animals were negative. In sexually plastic intersex animals, removal of the androgenic gland results in vitellogenin transcription, indicating that the gene is negatively regulated by the androgenic gland.